Effect of long-term around the clock illumination on rabbit serum phospholipids

This study was designed to investigate the effects of light on pineal gland function and phospholipids contents in the blood serum of rabbits. The level of melatonin in the blood serum was significantly reduced and endogenous rhythm of the melatonin secretion brok down under constant illumination conditions. Two-month treatment of rabbits with light led to a significant and short-term decrease of the phosphatidylethanolamine and phosphatidylinositol + phosphatidylserine levels in the serum. The contents of the other lipids under study (phosphatidylcholine, lysophosphatidylcholine and sphingomyelin) in blood increased later under 3-5 month-long constant illumination conditions. The results obtained demonstrate important role of pineal gland in the regulation of phospholipids metabolism.     

Pineal melatonin rhythms in the lizard Anolis carolinensis: I. Response to light and temperature cycles

Both light and temperature can influence the pineal's synthesis of the indoleamine melatonin. An investigation of the effects of light and temperature cycles on the pineal melatonin rhythm (PMR) showed the following: (1) Both daily light cycles and daily temperature cycles could entrain the PMR; melatonin levels peaked during the dark phase of a light-dark cycle or the cool phase of a temperature cycle. (2) The PMR could be entrained by a temperature cycle as low as 2 degrees C in amplitude in lizards held in constant light or constant darkness. (3) The length of the photoperiod or thermoperiod affected the phase, amplitude, or duration of the PMR. (4) When presented together, the effects of light and temperature cycles on the PMR depended on the phase relationship between the light and temperature cycles, as well as on the strength of the entraining stimuli, such as the amplitude of the temperature cycle. (5) Exposure to a constant cold temperature (10 degrees C) eliminated the PMR, yet a rhythm could still be expressed under a 24-hr temperature cycle (32 degrees C/10 degrees C), and the rhythm peaked during the 10 degrees C phase of the cycle. (6) A 6-hr dark pulse presented during the day did not elicit a premature rise in melatonin levels. These studies show how environmental stimuli can control the pineal rhythm of melatonin synthesis and secretion. Previous studies have supported a model in which the lizard's pineal acts as a circadian pacemaker within a multioscillator circadian system, and have implicated melatonin as a hormone by which the pineal may communicate with the rest of the system. The lizard pineal, therefore, may act as a photo- and thermoendocrine transducer translating light and temperature information into an internal cue in the form of the PMR. The PMR, in turn, may control the phase and period of circadian clocks located elsewhere, insuring that the right internal events occur at the right time of day.     

Avian Melatonin Synthesis: Photic and Circadian Regulation of Serotonin N-Acetyltransferase mRNA in the Chicken Pineal Gland and Retina

Abstract: The circadian rhythms in melatonin production in the chicken pineal gland and retina reflect changes in the activity of serotonin N -acetyltransferase (arylalkylamine N -acetyltransferase; AA-NAT; EC 2.3.1.87). Here we determined that the chicken AA-NAT mRNA is detectable in follicular pineal cells and retinal photoreceptors and that it exhibits a circadian rhythm, with peak levels at night. AA-NAT mRNA was not detected in other tissues. The AA-NAT mRNA rhythm in the pineal gland and retina persists in constant darkness (DD) and constant lighting (LL). The amplitude of the pineal mRNA rhythm is not decreased in LL. Light appears to influence the phase of the clock driving the rhythm in pineal AA-NAT mRNA in two ways: The peak is delayed by ∼6 h in LL, and it is advanced by >4 h by a 6-h light pulse late in subjective night in DD. Nocturnal AA-NAT mRNA levels do not change during a 20-min exposure to light, whereas this treatment dramatically decreases AA-NAT activity. These observations suggest that the rhythmic changes in chicken pineal AA-NAT activity reflect, at least in part, clock-generated changes in mRNA levels. In contrast, changes in mRNA content are not involved in the rapid light-induced decrease in AA-NAT activity.     

Ontogeny of N-acetyltransferase activity rhythm in pineal gland of chick embryo

1. N-acetyltransferase was present in pineal glands of 14-day-old chick embryos though no rhythm either in LL, DD or LD 12:12 was observed in this age. 2. Daily rhythm in pineal NAT activity was found in 18-day-old embryos incubated under LD 12:12 and LD 16:8 but no NAT rhythm was detected in DD or LL. 3. NAT rhythm persists for 2 days in constant darkness and it may be circadian in nature. 4. Presence of melatonin (85 +/- 8 pg/mg tissue) was detected in pineals of 18-day-old chick embryos.     

Day-Night Rhythm of Rat Pineal Tyrosine Hydroxylase Activity as Determined by HPLC with Amperometric Detection

Tyrosine hydroxylase activity in the rat pineal gland was measured by means of HPLC determination of the amount of L-3,4-dihydroxyphenylalanine formed. Enzyme activity showed a clear day-night rhythm, paralleling that of plasma melatonin levels in the same animals, with values being high during the dark period apparently because of changes in Vmax. In animals maintained under constant illumination for 3 days, tyrosine hydroxylase activity and plasma melatonin level rhythms were completely abolished, a result indicating that both are under photoperiodic control.     

Increase in testosterone sensitivity induced by constant light in relation to melatonin injections in rats.

In this experiment we investigated whether the lack of the nocturnal melatonin peak under constant light would cause an increase in testosterone sensitivity. Castrated rats were kept under periodic or constant light for one week. They received a daily injection of vehicle, testosterone propionate (125 micrograms), melatonin (50 micrograms) or testosterone plus melatonin (125 micrograms + 50 micrograms). Serum and pituitary gonadotrophins and pineal melatonin were measured at the end of the experiment. Under constant light, testosterone injections reduced the serum luteinizing hormone concentration in castrated rats to that in intact rats, but, under periodic light, the decrease was smaller. Melatonin did not reverse the stronger effect of testosterone under constant light. The serum melatonin peak produced by the exogenous melatonin injection had a higher amplitude, shorter duration and earlier appearance than the physiological melatonin peak. Exogenous melatonin did not modify the physiological melatonin secretion, measured either as serum melatonin concentration or pineal melatonin content on the consecutive day. We conclude that the increase in testosterone negative feedback sensitivity of castrated rats under constant light was not due to the absence of the nocturnal melatonin pulse.     

Pineal melatonin rhythms in the lizardAnolis carolinensis: effects of light and temperature cycles

Summary Pineal and ocular melatonin was assessed, over 24 h periods, in male lizards (Anolis carolinensis) entrained to 24 h light-dark (LD) cycles and a constant 32 C, and in lizards entrained to both 24 h LD cycles and 24 h temperature cycles (32 C/20 C). At a constant temperature, the duration of the photoperiod has a profound effect on the duration, amplitude, and phase of the pineal melatonin rhythm (Fig. 1). The pineal melatonin rhythm under cyclic temperature peaks during the cool (20 C) phase of the cycle regardless of whether or not the cool phase occurs during the light or dark phase of a LD 1212 cycle (Fig. 3). Under a temperature cycle and constant dim illumination, a pineal melatonin rhythm is observed which peaks during the cool phase of the temperature cycle, but the amplitude of the rhythm is depressed relative to that observed under LD (Fig. 2). Illumination up to 2 h in duration does not suppress the nocturnal melatonin peak in theAnolis pineal (Fig. 4). No melatonin rhythm was observed in the eyes ofAnolis under either 24 h LD cycles and a constant temperature (Fig. 1), or under simultaneous light and temperature cycles (Fig. 3). Ocular melatonin content was, in all cases, either very low or non-detectable.Abbreviations HIOMT hydroxyindole-O-methyltransferase - NAT N-acetyltransferase     

Daily and circadian variations of the pineal and ocular melatonin contents and their contributions to the circulating melatonin in the Japanese newt, Cynops pyrrhogaster

Daily and circadian variations of melatonin contents in the diencephalic region containing the pineal organ, the lateral eyes, and plasma were studied in a urodele amphibian, the Japanese newt (Cynops pyrrhogaster), to investigate the possible roles of melatonin in the circadian system. Melatonin levels in the pineal region and the lateral eyes exhibited daily variations with higher levels during the dark phase than during the light phase under a light-dark cycle of 12 h light and 12 h darkness (LD12:12). These rhythms persisted even under constant darkness but the phase of the rhythm was different from each other. Melatonin levels in the plasma also exhibited significant day-night changes with higher values at mid-dark than at mid-light under LD 12:12. The day-night changes in plasma melatonin levels were abolished in the pinealectomized (Px), ophthalmectomized (Ex), and Px+Ex newts but not in the sham-operated newts. These results indicate that in the Japanese newts, melatonin production in the pineal organ and the lateral eyes were regulated by both environmental light-dark cycles and endogenous circadian clocks, probably located in the pineal organ and the retina, respectively, and that both the pineal organ and the lateral eyes are required to maintain the daily variations of circulating melatonin levels.     

Circadian locomotor rhythms in the desert iguana I. The role of the eyes and the pineal

Summary The pineal and the eyes are known to be important components in the circadian system of some species of lizards; their effects may be mediated by the hormone melatonin. We examined the role played by these structures in the desert iguana (Dipsosaurus dorsalis). Surgical removal of the pineal had no effect on circadian locomotor rhythms, even though this procedure abolished the circadian rhythm of melatonin in the blood. Furthermore, when the isolated pineal of Dipsosaurus was studied in organ culture, it showed no circadian rhythm of melatonin secretion, as do pineals of some other lizard species, although it did produce large quantities of this hormone. Bilateral ocular enucleation had only small effects on the freerunning period of locomotor rhythms, without affecting melatonin levels in the blood. Behavioral circadian rhythms persisted in desert iguanas subjected to both enucleation and pinealectomy. These data suggest that neither the pineal nor the eyes are central components of the circadian pacemaking system in Dipsosaurus, nor is melatonin critically involved in maintaining its organization.Abbreviations CT circadian time - ZT zeitgeber time - LL constant light - LD light-dark cycle - DD constant darkness - freerunning circadian period     

Melatonin rhythms in the Australian freshwater crocodile (Crocodylus johnstoni): a reptile lacking a pineal complex?

The vertebrate pineal gland is the primary source of melatonin, the rhythmic secretion of which is influenced by environmental light and temperature, thereby providing animals with information about seasonally changing photoperiod and thermoperiod. Although pineal glands are present in the majority of vertebrate species, a discrete organ is reported to be absent in the Crocodilia. However, if the melatonin rhythm is crucial to the survival of the organism, it would be expected that the rhythm would be present in crocodiles. In the present study, we measured blood plasma melatonin over a 30-h period in aestivating Australian freshwater crocodiles (Crocodylus johnstoni) in their natural habitat at the end of the dry season (November) and found no discernible melatonin rhythm. However, another group of captive-reared C. johnstoni, maintained under natural light and temperature cycles and sampled in the early dry season (June) showed a clear melatonin rhythm. These results suggest that there is either an extrapineal source of melatonin in this crocodile species or that there is melatonin producing tissue elsewhere which heretofore has not been discovered. Further studies are needed to determine why the melatonin rhythm is intermittently expressed and whether this may be related to seasonal changes in the expression of the rhythm linked to tropical environments.     

Turkey retina and pineal gland differentially respond to constant environment

Dynamics of rhythmic oscillations in the activity of arylalkylamine N-acetyltransferase (AA-NAT, the penultimate and key regulatory enzyme in melatonin biosynthesis) were examined in the retina and pineal gland of turkeys maintained for 7 days in the environment without daily light-dark (LD) changes, namely constant darkness (DD) or continuous light (LL). The two tissues differentially responded to constant environment. In the retina, a circadian AA-NAT activity rhythm disappeared after 5 days of DD, while in the pineal gland it persisted for the whole experiment. No circadian rhythm was observed in the retinas of turkeys exposed to LL, although rhythmic oscillations in both AA-NAT and melatonin content were found in the pineal glands. Both tissues required one or two cycles of the re-installed LD for the full recovery of the high-amplitude AA-NAT rhythm suppressed under constant conditions. It is suggested that the retina of turkey is less able to maintain rhythmicity in constant environment and is more sensitive to changes in the environmental lighting conditions than the pineal gland. Our results indicate that, in contrast to mammals, pineal glands of light-exposed galliformes maintain the limited capacity to rhythmically produce melatonin.     

Development of the circadian melatonin rhythm and the effect of PACAP on melatonin release in the embryonic chicken pineal gland. An in vitro study

Pituitary adenylate cyclase activating polypeptide (PACAP) has been shown to participate in modulation of circadian rhythm and to stimulate melatonin (MT) secretion in both the rat and chicken pineal glands. In contrast to mammals, the main regulator of circadian rhythm in birds is the pineal gland, which begins its rhythmic MT production already during embryonic life. In the present study, we investigated the development of MT secretion in explanted embryonic chicken pineals and their responsiveness to PACAP in a perifusion system. Our results show that: (1) the circadian clock and/or the intracellular signal transduction system connecting the clock to MT synthesizing apparatus develop between the embryonic days 16-18 (E16-18), even in vitro. (2) Exposure of the embryonic chicken pineal gland to PACAP induces transitory increase in MT secretion but does not induce visible phase shift in the circadian rhythm. (3) Cyclic AMP (cAMP) efflux also responds to PACAP at or before day E13 in embryonic chicken pineal gland in vitro.     

The avian pineal gland

The pineal gland plays a key role in the control of the daily and seasonal rhythms in most vertebrate species. In mammals, rhythmic melatonin (MT) release from the pineal gland is controlled by the suprachiasmatic nucleus via the sympathetic nervous system. In most non-mammalian species, including birds, the pineal gland contains a self-sustained circadian oscillator and several input channels to synchronize the clock, including direct light sensitivity. Avian pineal glands maintain rhythmic activity for days under in vitro conditions. Several physical (light, temperature, and magnetic field) and biochemical (Vasoactive intestinal polypeptide (VIP), norepinephrine, PACAP, etc.) input channels, influencing release of melatonin are also functional in vitro, rendering the explanted avian pineal an excellent model to study the circadian biological clock. Using a perifusion system, we here report that the phase of the circadian melatonin rhythm of the explanted chicken pineal gland can be entrained easily to photoperiods whose length approximates 24 h, even if the light period is extremely short, i.e., 3L:21D. When the length of the photoperiod significantly differs from 24 h, the endogenous MT rhythm becomes distorted and does not follow the light-dark cycle. When explanted chicken pineal fragments were exposed to various drugs targeting specific components of intracellular signal transduction cascades, only those affecting the cAMP-protein kinase-A system modified the MT release temporarily without phase-shifting the rhythm in MT release. The potential role of cGMP remains to be investigated.     

The Avian Pineal Gland

The pineal gland plays a key role in the control of the daily and seasonal rhythms in most vertebrate species. In mammals, rhythmic melatonin (MT) release from the pineal gland is controlled by the suprachiasmatic nucleus via the sympathetic nervous system. In most non‐mammalian species, including birds, the pineal gland contains a self‐sustained circadian oscillator and several input channels to synchronize the clock, including direct light sensitivity. Avian pineal glands maintain rhythmic activity for days under in vitro conditions. Several physical (light, temperature, and magnetic field) and biochemical (Vasoactive intestinal polypeptide (VIP), norepinephrine, PACAP, etc.) input channels, influencing release of melatonin are also functional in vitro, rendering the explanted avian pineal an excellent model to study the circadian biological clock. Using a perifusion system, we here report that the phase of the circadian melatonin rhythm of the explanted chicken pineal gland can be entrained easily to photoperiods whose length approximates 24 h, even if the light period is extremely short, i.e., 3L:21D. When the length of the photoperiod significantly differs from 24 h, the endogenous MT rhythm becomes distorted and does not follow the light‐dark cycle. When explanted chicken pineal fragments were exposed to various drugs targeting specific components of intracellular signal transduction cascades, only those affecting the cAMP‐protein kinase‐A system modified the MT release temporarily without phase‐shifting the rhythm in MT release. The potential role of cGMP remains to be investigated.     

Arrest of the circadian pacemaker driving the pineal melatonin rhythm in hibernating golden hamsters, Mesocricetus auratus

Pineal melatonin rhythm in golden hamsters was abolished during hibernation. After arousal in darkness, pineal melatonin increased rapidly regardless of whether the arousal was induced during the day or at night. Rapid increase of pineal melatonin after arousal was markedly diminished in animals exposed to light. In hamsters aroused at midnight, the melatonin rhythm in constant darkness ran with the reversed phase relative to hamsters aroused at noon. Since after arousal the melatonin rhythm obviously starts anew from the same phase, we conclude that the circadian pacemaker driving the rhythm might be arrested during hibernation at the day-time phase.     

The effect of different wavelengths of light during incubation on the development of rhythmic pineal melatonin biosynthesis in chick embryos

Rhythmic pineal melatonin biosynthesis develops in chick embryos incubated under a light (L)-dark (D) cycle of polychromatic white light. The spectral sensitivity of the embryonic pineal gland is not known and was investigated in this study. Broiler breeder eggs (Ross 308, n=450) were incubated under white, red, green or blue light under the 12L : 12D cycle. Melatonin was measured in extracts of pineal glands by radioimmunoassay. The daily rhythm of pineal melatonin levels in 20-day-old chick embryos was confirmed during the final stages of embryonic life under all four wavelengths of light with expected higher concentrations during dark- than light-times. The highest pineal melatonin levels were determined in chick embryos incubated under red and white light and lower levels under green light. The incubation under blue light resulted in the lowest melatonin biosynthesis. Pineal melatonin concentrations increased substantially on post-hatching day two compared with pre-hatching levels and we did not find differences between birds incubated and kept in either white or green light. Our results demonstrate a selective sensitivity of the chick embryo pineal gland to different wavelengths of light. Rhythmic melatonin production is suggested as a possible mechanism, which transfers information about the quality of ambient light to the developing avian embryo.     

Photoperiod affects amplitude but not duration of in vitro melatonin production in the ruin lizard (Podarcis sicula)

The pineal gland and its major output signal melatonin have been demonstrated to play a central role in the seasonal organization of the ruin lizard Podarcis sicula. Seasonal variations in the amplitude of the nocturnal melatonin signal, with high values in spring as compared to low values in summer and autumn, have been found in vivo. The authors examined whether the pineal gland of the ruin lizard contains autonomous circadian oscillators controlling melatonin synthesis and whether previously described seasonal variations of in vivo melatonin production can also be found in isolated cultured pineal glands obtained from ruin lizards in summer and winter. In vitro melatonin release from isolated pineal glands of the ruin lizard persisted for 4 days in constant conditions. Cultured explanted pineal glands obtained from animals in winter and summer showed similar circadian rhythms of melatonin release, characterized by damping of the amplitude of the melatonin rhythm. Although different photoperiodic conditions were imposed on ruin lizards before explantation of pineal glands, the authors did not find any indication for corresponding differences in the duration of elevated melatonin in vitro. Differences were found in the amplitude of in vitro melatonin production in light/dark conditions and, to a lesser degree, in constant conditions. The presence of a circadian melatonin rhythm in vitro in winter, although such a rhythm is absent in vivo in winter, suggests that pineal melatonin production is influenced by an extrapineal oscillator in the intact animal that may either positively or negatively modulate melatonin production in summer and winter, respectively.     

Effect of long-term exposure to constant dim light on the circadian system of rats

The newly discovered multi-oscillatory nature of the mammalian circadian clock system and the cloning of the genes involved in the molecular mechanism that generates circadian rhythmicity have opened new approaches for understanding how mammals are temporally organized and how the mammalian circadian system reacts to the lack of normal synchronization cues. In the present study we investigated the effects of long-term exposure to constant red dim light on the pattern of the expression of Period 1 in the suprachiasmatic nuclei of the hypothalamus and of Arylalkylamine N-acetyltransferase(Aa-nat) in the retina and pineal gland. Our data demonstrate that Period 1 mRNA expression in the suprachiasmatic nuclei of the hypothalamus was not affected by exposure to constant red dim light for 60 days, whereas Aa-nat mRNA expression in the retina and in the pineal gland was significantly affected, since in some animals (20-30%) Aa-nat mRNA levels were found to be higher during the subjective day. A circadian rhythm of serum melatonin and locomotor activity was present in all the animals tested. In 4 animals serum melatonin levels were high during the subjective day. Our data suggest that long-term exposure to constant red dim light may induce desynchronization between the circadian rhythm of locomotor activity and serum melatonin levels.