Human Retinal Light Sensitivity and Melatonin Rhythms Following Four Days in Near Darkness

The rods in the retina are responsible for night vision, whereas the cone system enables day vision. We studied whether rod function in humans exhibits an endogenous circadian rhythm and if changes occur in conditions of prolonged darkness. Seven healthy subjects (mean age±SD: 25.6±12.3 yr) completed a 4.5‐day protocol during which they were kept in complete darkness (days 1 and 4) and near darkness (<0.1 lux red light, days 2 and 3). Electroretinography (ERG) and saliva collections were done at intervals of at least 3 h for 27 h on days 1 and 4. Full‐field ERGs were recorded over 10 low‐intensity green light flashes known to test predominantly rod function. As a circadian marker, salivary melatonin concentration was measured by radioimmunoassay. The ERG data showed that rod responsiveness to light progressively diminished in darkness (significantly lower a‐ and b‐wave amplitudes, longer b‐wave implicit time). The decrease in amplitude (b‐wave) from day 1 to day 4 averaged 22±14%. After correction for the darkness‐related linear trend, the circadian variations in ERG indices were weak and usually non‐significant, with slightly higher responsiveness to light during the day than night. Rod sensitivity (by K index) tended to decrease. Strikingly, the overall amount of melatonin secretion (area under 24 h curve) also decreased from day 1 to day 4 by 33.1±18.9% (p=.017). The drift of the melatonin rhythm phase was within the normal range, less than 56 min over three days. There was no significant correlation between the changes in ERG responses and melatonin. In conclusion, scotopic retinal response to (low‐intensity) light and the amount of melatonin secreted are diminished when humans are kept in continuous darkness. Both processes may have a common underlying mechanism implicating a variety of neurochemicals known to be involved in the regulation of both photoreceptor and pineal gland function.     

Human cone light sensitivity and melatonin rhythms following 24-hour continuous illumination

This study investigates the possibility of an endogenous circadian rhythm in retinal cone function in humans. A full-field cone electroretinogram (ERG) was performed every 2 h for 24 h under continuous rod-saturating ambient white light (53 ± 30 lux; pupils dilated) in nine healthy subjects. Distinct circadian variations were superimposed upon a gradual decrease in cone responsiveness to light, demonstrated most reliably in the implicit times of b-wave and oscillatory potentials, and to a lesser extent in amplitude and a-wave implicit times. After mathematical correction of the linear trend, the cone response was found to be greatest around 20:00 h and least around 06:00 h. The phase of the ERG circadian rhythm was not synchronized with the phase of the salivary melatonin rhythm measured the previous evening. Melatonin levels measured under constant light on the day of ERG assessments were suppressed by 53% on average compared to melatonin profiles obtained previously under near-total darkness in seven participants. The progressive decline in cone responsiveness to light over the 24 h may reflect an adaptation of the cone-driven retinal system to constant light, although the mechanism is unclear. The endogenous rhythm of cone responsiveness to light may be used as an additional index of central or retinal circadian clock time.     

Comparative study of the rod and cone contributions to the generation of b-wave ERG and tectal evoked potential in the dark-adapted carp

Amplitudes and peak latencies as functions of wave length and monochromatic light intensity were investigated for b-wave ERG and tectal evoked potentials (EP) in the dark-adapted carp (Cyprinus carpio L). It was found, that independently of light intensity b-wave action spectra had one maximum in the medium wave band, corresponding to rod sensitivity area. For tectal EP, similar action spectra with maximum in the middle-wave were seen at low light intensity only. The b-wave amplitude growth was significant for the whole band of light intensities, and these changes were accompanied with a slight decrease in peak latency (to 50-100 ms). Tectal EP amplitude increased when low-intensity light was changed for medium intensity light and did not considerably increase to brighter light stimuli. However, tectal EP time latency significantly decreased (to 100-200 ms) during light intensity increasing. This differences show that retinal rod system, which in responsible for ERG b-wave in darkness, is not a key factor in the generation of tectal EP.     

正常猕猴与人的视网膜电图比较

目的比较正常猕猴与人视网膜电图异同,为进一步利用猕猴建立动物模型研究视网膜疾病打下基础。方法健康成年猕猴7只（14只眼）与8例（16只眼）正常人进行视网膜电图检测,对两者Rod-ERG中的b波,Max-ERG的a、b波,Cone-ERG的b波峰时值及波幅和OPs的O2值,Flicker-ERG的P值进行统计学检验。结果猕猴与人的视网膜电图波形结果较为相似,各指标与人的结果相比,潜伏期短,幅值低,但Cone-ERG和Flicker-ERG两者幅值差异不具有统计学意义。结论视网膜电图检测从功能上证明猕猴较其他常用实验动物更接近人,尤其表现在视锥细胞及黄斑区功能,可用作人类视网膜疾病尤其是黄斑区病变的良好动物模型。     

Human Cone Light Sensitivity and Melatonin Rhythms Following 24-hour Continuous Illumination

This study investigates the possibility of an endogenous circadian rhythm in retinal cone function in humans. A full-field cone electroretinogram (ERG) was performed every 2?h for 24?h under continuous rod-saturating ambient white light (53 ±?30 lux; pupils dilated) in nine healthy subjects. Distinct circadian variations were superimposed upon a gradual decrease in cone responsiveness to light, demonstrated most reliably in the implicit times of b-wave and oscillatory potentials, and to a lesser extent in amplitude and a-wave implicit times. After mathematical correction of the linear trend, the cone response was found to be greatest around 20:00?h and least around 06:00?h. The phase of the ERG circadian rhythm was not synchronized with the phase of the salivary melatonin rhythm measured the previous evening. Melatonin levels measured under constant light on the day of ERG assessments were suppressed by 53% on average compared to melatonin profiles obtained previously under near-total darkness in seven participants. The progressive decline in cone responsiveness to light over the 24?h may reflect an adaptation of the cone-driven retinal system to constant light, although the mechanism is unclear. The endogenous rhythm of cone responsiveness to light may be used as an additional index of central or retinal circadian clock time. (Author correspondence: kvdani@mail.ru)     

Transient Fluctuation of Serum Melatonin Rhythm is Suppressed Centrally by Vitamin B

Vitamin B₁₂ has been reported to improve sleep-wake rhythm disorders. Although the mechanism is still unclear, a change in the sensitivity of the circadian clock system to photic input is thought to be a possible mechanism of the effect. In this study, the effect of the vitamin B₁₂ on the circadian aspect of the electroretinogram (ERG) and serum melatonin level was analyzed in rats. Vitamin B₁₂, α-(5,6-dimethylbenzimidazolyl)-co-methyl-cobamide was daily administrated subcutaneously for 8 weeks to adult male Wister rats in the experimental group, and saline was given to the control group. The ERGs were recorded under dark adaptation during the night and day, and under light adaptation (0.1 lux) during the night. Blood was drawn before and after ERG recording. The amplitudes of the a-wave, fc-wave, and trough-to-peak of both waves and latencies of ERG were analyzed following various exposures to stimuli of light intensity. These parameters in the group treated with vitamin B₁₂ showed similar characteristics to the control group, and no significant difference was observed between the two groups. The melatonin levels of both groups before the measurement of ERG were similar under each measurement condition. The elevated serum melatonin concentration in the control group under dark adaptation at night was suppressed after the series of 10-msec light stimuli used for measurement of ERG. However, this suppressing effect of light pulses on melatonin level was significantly inhibited in the group treated with vitamin B₁₂. Under light adaptation during the night and under dark adaptation during the day, melatonin levels after the measurement of ERG were not different between the groups. From these results, it is suggested that vitamin B₁₂ is effective in suppressing melatonin rhythm disturbances introduced by transient light stimulation, and it affects the site more central than the retinal level. (Chronobiology International, 14(6), 549–560, 1997)     

Rod-type transducin alpha-subunit mediates a phototransduction pathway in the chicken pineal gland

The chicken pineal gland is a photosensitive neuroendocrine organ producing melatonin in circadian clock-regulated and light-sensitive manners. To understand the relationship between the photoreceptive molecule pinopsin and the light-dependent melatonin suppression that is sensitive to pertussis toxin treatment, we have searched for pertussis toxin-sensitive G protein alpha-subunits expressed in the chicken pineal gland. Here we report the cDNA cloning of the pineal transducin alpha-subunit (Gtalpha), which is highly homologous to human retinal rod cell-specific Gt(1)alpha. Concurrent cDNA cloning of chicken retinal Gt(1)alpha and Gt(2)alpha (rod and cone cell-specific alpha-subunits of transducin, respectively) revealed that the chicken pineal Gtalpha is identical to the retinal Gt(1)alpha. Double-immunostaining analysis of the chicken pineal sections localized Gt(1)alpha-immunoreactivity in the rudimentary outer segments of both follicular and parafollicular pinealocytes that were immunopositive to anti-pinopsin antibody. To examine whether pineal Gt(1)alpha is involved in the pineal phototransduction pathway, trypsin protection assay was applied for detecting the conversion of GDP-bound Gt(1)alpha into the guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS)-bound form in the pineal membrane homogenate. It was clearly demonstrated that the pineal Gt(1)alpha is activated in a light-dependent manner in the presence of GTPgammaS. These data together suggest strongly that pineal Gt(1)alpha mediates the phototransduction pathway triggered by pinopsin in the chicken pinealocytes.     

Light intensities required to suppress nocturnal melatonin secretion in albino and pigmented rats

Sprague-Dawley albino rats or Long-Evans pigmented rats were exposed during the dark phase of the daily light:dark cycle to various intensities of a sunlight-stimulating white fluorescent light (0.022, 0.044, 0.110, 0.220, 0.440 or 2.200 μW/cm²) for 30 min; pineal glands and trunk blood samples were then collected and assayed for melatonin by radioimmunoassay. Albino rats exposed to irradiances of 0.110 μW/cm² or less had pineal melatonin levels that were not significantly different from those of unexposed animals; higher irradiances significantly (P < 0.001) reduced melatonin levels. In contrast, as little as 0.022 μW/cm² significantly (P < 0.02) reduced pineal and serum melatonin levels in the pigmented rats. These results suggest that something other than the simple presence or absence of eye pigmentation is the critical factor in determining the sensitivity of the rat's pineal to retinal-mediated photic suppression of melatonin synthesis.     

Age-related differences in serum melatonin and pineal NAT activity and in the response of rat pineal to a 50-Hz magnetic field.

In a previous study we have shown that exposure to a 50-Hz sinusoidal magnetic field decreased serum melatonin concentration and pineal enzyme activities in young rats (9 weeks). In the present study we looked for the effect of a magnetic field of 100 microT on serum melatonin and pineal NAT activity in aged rats and compared them to young rats. We hypothesized that aging may change sensitivity of rats to a magnetic field. Two groups of Wistar male rats [aged rats (23 months) and young rats (9 weeks)] were exposed to 50-Hz magnetic fields of 100 microT for one week (18h/day). The animals were kept under a standard 12:12 light: dark cycle with a temperature of 25 degrees C and a relative humidity of 45 to 50%. Control (sham-exposed) animals were kept in a similar environment but without exposure to a magnetic field. The animals were sacrificed under red dim light. Serum melatonin concentration and pineal N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) activities were studied. Our results showed that sinusoidal magnetic fields altered the production of melatonin (28% decrease; P <0.05) through an inhibition of pineal NAT activity (52% decrease; P <0.05) in the young rats whereas no effect was observed in aged ones. On the other hand, when comparing data from control animals between young and aged rats, we observed that serum melatonin level and NAT activity, but not HIOMT activity, decreased in aged rats (decrease by about 38% and 36% respectively). Our data strongly suggest that old rats are insensitive to the magnetic field.     

Effect of Lithium on the Rhythms of Melatonin in the Pineal Gland, Serum and Retina of Viscacha (Lagostomus maximus maximus)

The aim of this work was to describe the pineal, serum and retinal melatonin daily rhythms in viscacha (Lagostomus maximus maximus), a nocturnal and subterranean rodent. A daily rhythm with a nocturnal peak and an increase during the light phase was found in all cases, with the seric rhythm being delayed with respect to the pineal one. Chronic lithium administration to viscachas suppressed the diurnal increase of pineal melatonin, abolished the retinal rhythm and had no effect on seric melatonin.     

Photic and circadian regulation of melatonin production in the Mozambique tilapia Oreochromis mossambicus

Diverse circadian systems related to phylogeny and ecological adaptive strategies are proposed in teleosts. Recently, retinal photoreception was reported to be important for the circadian pacemaking activities of the Nile tilapia Oreochromis niloticus. We aimed to confirm the photic and circadian responsiveness of its close relative-the Mozambique tilapia O. mossambicus. Melatonin production in cannulated or ophthalmectomized fish and its secretion from cultured pineal glands were examined under several light regimes. Melatonin production in the cannulated tilapias was measured at 3-h intervals; it fluctuated daily, with a nocturnal increase and a diurnal decrease. Exposing the cannulated fish to several light intensities (1500-0.1 lx) and to natural light (0.1 and 0.3 lx) suppressed melatonin levels within 30 min. Static pineal gland culture under light-dark and reverse light-dark cycles revealed that melatonin synthesis increased during the dark periods. Rhythmic melatonin synthesis disappeared on pineal gland culture under constant dark and light conditions. After ophthalmectomy, plasma melatonin levels did not vary with light-dark cycles. These results suggest that (1) Mozambique tilapias possess strong photic responsiveness, (2) their pineal glands are sensitive to light but lack circadian pacemaker activity, and (3) they require lateral eyes for rhythmic melatonin secretion from the pineal gland.     

视网膜电图振荡电位参数的稳定性

用系列强度１１档刺激闪光记录２０例３０只眼的视网膜电图（ＥＲＧ）振荡电位（ＯＰ）,研究ＯＰ的光强特性和参数稳定性。结果表明,ＯＰ各子波峰时和刺激光强呈指数曲线相关,相关性很高;而振幅和光强的函数关系的变异很大,相关性较低,不同对象间ＯＰ各子波振幅的变异系数较大,而峰时的变异系数较小,ＯＰ的峰时改变比振幅改变更为稳定。     

INHERITED RETINAL DYSTROPHY IN THE RAT

Retinal dystrophies, known in man, dog, mouse, and rat, involve progressive loss of photoreceptor cells with onset during or soon after the developmental period. Functional (electroretinogram), chemical (rhodopsin analyses) and morphological (light and electron microscopy) data obtained in the rat indicated two main processes: (a) overproduction of rhodopsin and an associated abnormal lamellar tissue component, (b) progressive loss of photoreceptor cells. The first abnormality recognized was the appearance of swirling sheets or bundles of extracellular lamellae between normally developing retinal rods and pigment epithelium; membrane thickness and spacing resembled that in normal outer segments. Rhodopsin content reached twice normal values, was present in both rods and extracellular lamellae, and was qualitatively normal, judged by absorption maximum and products of bleaching. Photoreceptors attained virtually adult form and ERG function. Then rod inner segments and nuclei began degenerating; the ERG lost sensitivity and showed selective depression of the a-wave at high luminances. Outer segments and lamellae gradually degenerated and rhodopsin content decreased. No phagocytosis was seen, though pigment cells partially dedifferentiated and many migrated through the outer segment-debris zone toward the retina. Eventually photoreceptor cells and the b-wave of the ERG entirely disappeared. Rats kept in darkness retained electrical activity, rhodopsin content, rod structure, and extracellular lamellae longer than litter mates in light.     

Negative correlation of age and the levels of pineal melatonin, pineal N-acetylserotonin, and serum melatonin in male rats

Pineal concentrations of N-acetylserotonin and melatonin and serum levels of melatonin were studied in 3-wk-old (prepubertal), 8-wk-old (adult), and 17-mo-old (senile) male rats. They were adapted to a photoperiod of 12 h light/12 h darkness for a minimum of 1 wk and killed at mid-light and mid-dark. Melatonin and N-acetylserotonin were determined by radioimmunoassay. The concentrations of pineal N-acetylserotonin and melatonin were high in the dark period and low in the light period. Statistical analysis indicated that pineal N-acetylserotonin and melatonin levels per 100 gm body weight declined with age. Similarly, serum melatonin demonstrated diurnal changes in all the age groups studied. In addition, there was a significant reduction in the levels of serum melatonin with age. The parallel patterns of decrease in pineal and serum melatonin levels with age suggest a decline in pineal secretion of melatonin in the older animals.     

The Effect on Body Temperature and Melatonin of A 39-H Constant Routine with Two Different Light Levels at Nighttime

Eight healthy subjects were studied during 39-h spans (from 07:00 on one day until 22:00 the second) in which they remained awake. During one experiment, subjects were exposed to 100 lux of light between 18:00 and 8:00, and during a second experiment, they were exposed to 1000 lux during the same time span. Throughout the daytime period, they were exposed to normal daylight (1500 lux or more). The nighttime 1000-lux light treatment suppressed the melatonin metabolite aMT6s, while the 100 lux treatment did not. On the treatment day, the 1000 lux, in comparison to the 100 lux, light treatment resulted in both an elevated temperature minimum and a delay in its clock-time occurrence overnight. No real circadian phase shift in the temperature, urinary melatonin, or Cortisol rhythms was detected after light treatment. This study confirmed that nocturnal exposure to lower light intensities is capable of modifying circadian variables more than previously estimated. The immediate effects of all-night light treatment are essentially not different from those of evening light. This may be important if bright light is used to improve alertness of night workers. Whether subsequent daytime alertness and sleep recovery are affected by the protocol used in our study remains to be determined.     

Circadian regulation of pineal melatonin and reproduction in the Djungarian hamster

Gonadal state, pineal melatonin rhythms, and locomotor activity rhythms were examined in juvenile male Djungarian hamsters exposed to non-24-hr light cycles ("T-cycles") or to full photoperiods. At the end of 1 month, hamsters exposed to a 1-hr pulse of light every 24.33 hr (T 24.33) exhibited small testes, whereas those receiving the same amount of light every 24.78 hr (T 24.78) displayed stimulated gonads, ten-fold larger in size. Accompanying the nonstimulatory effect of the T 24.33 cycle were nocturnal peaks in both pineal melatonin content and serum melatonin concentration which were longer by approximately 4 hr than those observed on the photostimulatory T 24.78 cycle. Exposure to an intermediate-length T-cycle (T 24.53) resulted in a mixed gonadal response and in pineal and serum melatonin peaks of intermediate duration. Wheel-running activity was entrained to the T-cycles such that light was present only near the beginning of the subjective night, its phase (relative to activity onset) differing only slightly among T-cycle groups. Hence the durational differences observed in the melatonin peaks were apparently not due to the acute suppressive or phase-advancing effects of morning light on melatonin biosynthesis, but were rather the result of differences in the endogenous control of pineal activity by the circadian pacemaker system. While no strong correlation was detected between gonadal state and the phase of locomotor activity onset relative to the light pulse, a significant correlation was observed between gonadal state and the duration of daily locomotor activity (alpha). These data were compared to similar measures obtained from hamsters exposed to long-versus short-day full photoperiods (LD 16:8 vs. LD 10:14). In summary, the results of this study indicate involvement of the circadian pacemaker system of Djungarian hamsters in the control of pineal melatonin synthesis and secretion, and in photoperiodic time measurement. Furthermore, these data strengthen the hypothesis that it is the duration of nocturnal pineal melatonin secretion that is the critical feature of this neuroendocrine gland's photoperiodic signal.     

Ex vivo studies on the acute and chronic effects of DHEA and DHEA-sulfate on melatonin synthesis in young- and old-rat pineal glands

This study investigates the effects of acute and chronic injections of the neurosteroid dehydroepiandrosterone (DHEA) and its sulfate DHEA-S on pineal gland melatonin synthesis. Pineal melatonin production and plasma melatonin levels were investigated in young (9-week-old) and old (27-month-old) male Wistar rats. DHEA or DHEA-S have been administered acutely in a single intraperitoneal injection at a dosage of 50, 250, or 500 microg per animal, or on a long-term basis, i.e., for 8 days at a dosage of 100 microg per animal, 1 h before the onset of darkness. DHEA, at a dose of 50, 250, or 500 microg per animal, administered acutely to rats had no significant effects on pineal melatonin production whatever the age of the animals. In contrast, 500 microg DHEA-S induced a significant increase in the pineal melatonin content (15% in young animals and 35% in old animals) and the activity of N-acetyltransferase, the rate-limiting enzyme for melatonin synthesis in the pineal gland, (40% in young animals and 20% in old animals), without altering the activity of hydroxyindole-O-methyltransferase whatever the age of the animals. At lower concentrations (50 or 250 microg) DHEA-S had no effect on pineal melatonin production regardless of the age of the rats. Chronic injection of DHEA or DHEA-S at a dose of 100 microg had no effect on pineal melatonin or NAT and HIOMT activities in the two age groups. This work shows that DHEA-S (and not DHEA) is able, at pharmacological concentrations, to stimulate melatonin production by rat pineal glands regardless of the age of the animals.     

Effects of melatonin and 6-methoxy-tetrahydro-beta-carboline in light induced retinal damage: a computerized morphometric method

The effects of melatonin and a related 5-methoxy-indole, 6-methoxy-1,2,3,4-tetrahydro-beta-carboline (6-MeO-THBC) were investigated in rats on the development of retinal degeneration in presence of high intensity illumination (HII). A morphometric method is used in which the degree of degeneration was evaluated by a computer-coupled graphical analyzer. Instead of measuring individual thicknesses of different retinal layers at various loci we measured large areas of retinal light microscopic sections. Thus the influence of sporadic artefactual and other fluctuations in the thickness of various layers of the retina can be essentially reduced. Continuous light produced significant degeneration of the retina and the degree of degeneration was further increased by both studied compounds and even more by 6-MeO-THBC. The role of melatonin and 6-MeO-THBC in retinal physiology is discussed.     

Differences in Retinal Structure and Function between Aging Male and Female Sprague-Dawley Rats are Strongly Influenced by the Estrus Cycle

Purpose

Biological sex and age are considered as two important factors that may influence the function and structure of the retina, an effect that might be governed by sexual hormones such as estrogen. The purpose of this study was to delineate the influence that biological sex and age exert on the retinal function and structure of rodents and also clarify the effect that the estrus cycle might exert on the retinal function of female rats.

Method

The retinal function of 50 normal male and female albino Sprague-Dawley (SD) rats was investigated with the electroretinogram (ERG) at postnatal day (P) 30, 60, 100, 200, and 300 (n = 5–6 male and female rats/age). Following the ERG recording sessions, retinal histology was performed in both sexes. In parallel, the retinal function of premenopausal and menopausal female rats aged P540 were also compared.

Results

Sex and age-related changes in retinal structure and function were observed in our animal model. However, irrespective of age, no significant difference was observed in ERG and retinal histology obtained from male and female rats. Notwithstanding the above we did however notice that between P60 and P200 there was a gradual increase in ERG amplitudes of female rats compared to males. Furthermore, the ERG of premenopausal female rats aged 18 months old (P540) was larger compared to age-matched menopausal female rats as well as that of male rats.

Conclusion

Our results showed that biological sex and age can influence the retinal function and structure of albino SD rats. Furthermore, we showed that cycled female rats have better retinal function compared to the menopausal female rats suggesting a beneficial effect of the estrus cycle on the retinal function.