Photoperiodic control of rat pineal serotonin N-acetyl-transferase activity

Adult male albino rats were acclimated to constant light (light:dark-LD-24:0) or to darkness interrupted with brief periods of light at 6 h intervals (LD 1/4:5 3/4 X 4) concurrently with rats maintained in a LD 14:10 photoperiodic cycle. The activity and rhythmicity of pineal serotonin N-acetyltransferase (NAT) was examined at regular intervals for 24 hours in rats maintained in the experimental photoperiods and compared to pineal NAT activity and rhythmicity in rats maintained in the LD 14:10 photoperiod. The results indicate that constant light is capable of depressing nocturnal levels of rat pineal NAT and obliterating the pineal NAT rhythm. Likewise, rats subjected to darkness interrupted with brief periods of light at 6 h intervals experienced a similar response in pineal NAT activity to animals subjected to constant light, i.e., pineal NAT activity was persistently low and the rhythmicity was obliterated. The results are discussed relative to the hypothesis that the pineal NAT activity responds to an endogenous rhythm in photoperiodic time measurement. The evidence herein suggests that the time of occurrence of environmental light in the photoperiod is more important in determining pineal NAT activity and/or rhythmicity than is the total amount of darkness or the dark to light ratio to which animals may be subjected.     

Regulation by Light and Darkness of Melatonin Secretion from the Superfused Masu Salmon (Oncorhynchus masou) Pineal Organ

The pineal organ of masu salmon Oncorhynchus masou was maintained in a flow-through, whole-organ culture (superfusion) system and melatonin secretory profiles were determined at 15 °C under light-dark cycles of 12:12 h (LD 12:12) or the same in combination with constant darkness (DD) for 72 h. Under LD 12:12, superfused pineal organs showed a rhythmic melatonin secretion with high and low rates during the dark phase and the light phase, respectively. When the pineal organs maintained under LD 12:12 for 24 h were transferred to DD, melatonin secretion was consistently activated and no endogenous component was evident. When the pineal organs maintained under DD for 48 h were transferred to LD 12:12, melatonin secretion was reduced only during the light phase. These results indicate that melatonin secretion from the superfused pineal organ of masu salmon is regulated not by an intra-pineal circadian oscillator but by the environmental LD cycles, via local photoreceptors.     

Regulation by Light and Darkness of Melatonin Secretion from the Superfused Masu Salmon (Oncorhynchus masou) Pineal Organ

The pineal organ of masu salmon Oncorhynchus masou was maintained in a flow-through, whole-organ culture (superfusion) system and melatonin secretory profiles were determined at 15 °C under light-dark cycles of 12:12 h (LD 12:12) or the same in combination with constant darkness (DD) for 72 h. Under LD 12:12, superfused pineal organs showed a rhythmic melatonin secretion with high and low rates during the dark phase and the light phase, respectively. When the pineal organs maintained under LD 12:12 for 24 h were transferred to DD, melatonin secretion was consistently activated and no endogenous component was evident. When the pineal organs maintained under DD for 48 h were transferred to LD 12:12, melatonin secretion was reduced only during the light phase. These results indicate that melatonin secretion from the superfused pineal organ of masu salmon is regulated not by an intra-pineal circadian oscillator but by the environmental LD cycles, via local photoreceptors.     

Adrenal-mediated depression of N-acetyltransferase activity and melatonin levels in the rat pineal gland

N-acetyltransferase (NAT) is believed to be the rate-limiting enzyme in the synthesis of melatonin from serotonin in the pineal gland. Norepinephrine released from sympathetic nerve endings within the pineal gland stimulates NAT activity and, therefore, melatonin synthesis. When an animal is subjected to a stressful stimulus, it would be expected that the increase in plasma stimulus, it would be expected that the increase in plasma catecholamines originating from the adrenal medulla and/or the sympathetic nervous system would result in a stimulation of pineal NAT activity. Adult male rats were given a 1.5cc injection of physiological saline subcutaneously into the back leg. Compared to non-injected controls, animals stressed in this manner were shown to have significantly lower pineal melatonin content 10 min after the saline injection late in the light phase of the light/dark cycle (at 18.30 h-lights on at 07.00 h). To test this more thoroughly, a time course study was conducted during the dark phase (at 02.00 h-5 hours after lights out) when pineal NAT activity and melatonin levels are either increasing or elevated. NAT activity and melatonin levels in the pineal were significantly depressed in stressed animals as compared to controls by 10 min after the saline injection, and remained so until 60 min after injection. By 90 min they had returned to control values. In the next study the nighttime response of the pineal to stress was compared in intact and adrenalectomized rats. Adrenalectomy prevented the changes in NAT activity and melatonin content associated with the saline injection. Some factor, such as a catecholamine or corticosterone from the adrenal, seems to be eliciting the response in the pineal to the saline injection. It is not known if the factor is acting centrally or directly on the pineal gland.     

Developmental patterns of FSH and LH in female rats deprived of light before puberty.

Plasma FSH and LH levels were examined in female rats reared in the dark at different ages from birth until sexual maturation to investigate whether, and to what extent, external factors such as light, influence gonadotropin levels during development. Control animals were raised in diurnal lighting consisting of 12 hours of light and 12 hours of dark. Light deprivation did not eliminate the characteristic peak of gonadotropins seen in early postnatal development but significantly increased levels of FSH and slightly decreased levels of LH (except for a transient rise at day 12). Constant darkness tended to lower whole body, ovarian and pituitary weights but to increase pineal weight. Whereas the time of eye-opening was the same in control and light-deprived animals, puberty (as judged by vaginal opening and first ovulation) was delayed in animals raised in the dark. The data suggest that environmental light has a mediating action on patterns of gonadotropin release, particularly on FSH, during prepuberal development.     

Suppression of circadian rhythms of pineal and testicular hormones following lithium treatment in normal and reversed light-dark cycles, constant light and constant dark in rats

The aim of the current investigation was to study the effect of lithium on circadian rhythms of pineal - testicular hormones by quantitations of pineal and serum serotonin, N-acetylserotonin and melatonin, and serum testosterone at four time points (06.00, 12.00, 18.00 and 24.00) of a 24-hr period under normal photoperiod (L:D), reversed photoperiod (D:L), constant light (L:L) and constant dark phase (D:D) in rats. Circadian rhythms were observed in pineal hormones in all the combinations of photoperiodic regimens, except in constant light, and in testosterone levels in all the photoperiodic combinations. Pineal and serum N-acetylserotonin and melatonin levels were higher than serotonin at night (24.00 hr), in natural L:D cycle, in reversed L:D cycle or similar to normal L:D cycle in constant dark phase, without any change in constant light. In contrast, testosterone level was higher in light phase (12.00 hr through 18.00 hr) than in the dark phase (24.00 hr through 06.00 hr) in normal L:D cycle, in reversed L:D cycle, similar to normal L:D cycle in constant dark (D:D), and reversed to that of the normal L:D cycle in constant light (L:L). Lithium treatment (2 mEq/kg body weight daily for 15 days) suppressed the magnitude of circadian rhythms of pineal and serum serotonin, N-acetylserotonin and melatonin, and testosterone levels by decreasing their levels at four time points of a 24-hr period in natural L:D or reversed D:L cycle and in constant dark (D:D). Pineal indoleamine levels were reduced after lithium treatment even in constant light (L:L). Moreover, lithium abolished the melatonin rhythms in rats exposed to normal (L:D) and reversed L:D (D:L) cycles, and sustained the rhythms in constant dark. But testosterone rhythm was abolished after lithium treatment in normal (L:D)/reversed L:D (D:L) cycle or even in constant light/dark. The findings indicate that the circadian rhythm exists in pineal hormones in alternate light - dark cycle (L:D/D:L) and in constant dark (D:D), but was absent in constant light phase (L:L) in rats. Lithium not only suppresses the circadian rhythms of pineal hormones, but abolishes the pineal melatonin rhythm only in alternate light - dark cycles, but sustains it in constant dark. The testosterone rhythm is abolished after lithium treatment in alternate light - dark cycle and constant light/dark. It is suggested that (a) normal circadian rhythms of pineal hormones are regulated by pulse dark phase in normal rats, (b) lithium abolishes pineal hormonal rhythm only in pulse light but sustains it in constant dark phase, and (c) circadian testosterone rhythm occurs in both pulse light or pulse dark phase in normal rats, and lithium abolishes the rhythm in all the combinations of the photoperiod. The differential responses of circadian rhythms of pineal and testicular hormones to pulse light or pulse dark in normal and lithium recipients are discussed.     

Induction of c-fos protein-like immunoreactivity in the rat and hamster pineal gland after the onset of darkness

Induction of c-fos protein (FOS) after the onset of darkness was studied immunocytochemically in the rat and hamster pineal gland. The animals were kept on a 12:12 h light-dark cycle. Before the dark period no FOS staining was seen in either rat or hamster pineal cells. Five hours after the onset of darkness 342 +/- 18 pinealocytes/0.2 mm2 (mean +/- SD) displayed FOS-like immunoreactivity in the hamster pineal gland; in the rat pineal gland only 5 +/- 2 pinealocytes/0.2 mm2 showed a faint staining. Two hours later the density of FOS positive cells was decreased to 60 +/- 11/0.2 mm2 in the hamster but increased to 519 +/- 103/0.2 mm2 in the rat pineal gland. Three hours before the beginning of the light period no FOS positive cells were detected in either animal. Both the rat and hamster pineal gland showed a transient and temporally defined expression of c-fos protein in the middle of the dark period. This may be related to a more active functional state of pinealocytes, which is reflected in a peak of melatonin synthesis during the darkness.     

Electric field exposure alters serum melatonin but not pineal melatonin synthesis in male rats

Sprague-Dawley male rats, maintained in a 14:10 h light:dark cycl were exposed for 30 days (starting at 56 days of age) to a 65 kV/m, 60 Hz electric field or to a sham field for 20 h/day beginning at dark onset. Pineal N-acetyltransferase (NAT), hydroxy-indole-o-methyl transferase (HIOMT), and melatonin as well as serum melatonin were assayed. Preliminary data on unexposed animals indicated that samples obtained 4 h into the dark period would reveal either a phase delay or depression in circadian melatonin synthesis and secretion. Exposure to electric fields for 30 days did not alter the expected nighttime increase in pineal NAT, HIOMT, or melatonin. Serum melatonin levels were also increased at night, but the electric field-exposed animals had lower levels than the sham-exposed animals. Concurrent exposure to red light and the electric field or exposure to the electric field at a different time of the day-night period did not reduce melatonin synthesis. These data do not support the hypothesis that chronic electric field exposure reduces pineal melatonin synthesis in young adult male rats. However, serum melatonin levels were reduced by electric field exposure, suggesting the possibility that degradation or tissue uptake of melatonin is stimulated by exposure to electric fields. © 1994 Wiley-Liss, Inc.     

Effects of acute ethanol administration on nocturnal pineal serotonin N-acetyltransferase activity

The effect of acute ethanol administration on pineal serotonin N-acetyltransferase (NAT) activity, norepinephrine and indoleamine content was examined in male rats. When ethanol was administered in two equal doses (2 g/kg body weight) over a 4 hour period during the light phase, the nocturnal rise in NAT activity was delayed by seven hours. The nocturnal pineal norepinephrine content was not altered by ethanol except for a delay in the reduction of NE with the onset of the following light phase. Although ethanol treatment led to a significant reduction in nocturnal levels of pineal serotonin content, there was no significant effect upon pineal content of 5-hydroxyindoleacetic acid (5-HIAA). The data indicate that ethanol delays the onset of the rise of nocturnal pineal NAT activity.     

Diurnal and circadian variations in indole contents in the goose pineal gland

ABSTRACT

The diurnal and circadian profiles of pineal indoles, except melatonin, are poorly characterized in birds. Moreover, there are no data on the effect of sudden changes in the light–dark cycle on these profiles. Therefore, we investigated the diurnal (Experiment I) and circadian variation (Experiment II) of nine pineal indoles (tryptophan, 5-hydroxytryptophan, serotonin, N-acetylserotonin, melatonin, 5-hydroxyindole acetic acid, 5-methoxytryptophol, 5-methoxyindole acetic acid, 5-methoxytryptamine) in geese, as well as the changes in the profiles of these substances in geese subjected to a reversed light–dark cycle (Experiment III). For the first 12 weeks of life, all geese were kept under a diurnal cycle of 12 h of light and 12 h of darkness (12L:12D). In Experiment I (n = 48), they were kept under these conditions for another 14 days before being sacrificed at 2-h intervals for sampling of the pineal glands. In Experiment II, the geese (n = 48) were divided into three groups (12L:12D, 24L:0D, 0L:24D) for 10 days before sampling at 6-h intervals. In Experiment III, 24 geese were exposed to a reversed light–dark cycle before sampling at 14:00 and 02:00 on the first, second and third days after light–dark cycle reversal. To determine the content of the indoles in the goose pineals, HPLC with fluorescence detection was used. We found that, with the exception of tryptophan, all the investigated indoles showed statistically significant diurnal variation. When geese were kept in constant darkness, most of the indoles continued to show this variation, but when geese were kept in constant light, the indoles did not show significant variation. When the light–dark cycle was reversed (12L:12D to 12D:12L), the profiles of NAS, melatonin, 5-MTAM and 5-MTOL reflected the new cycle within 2 days. The content of serotonin in geese in 12L:12D was higher than that observed in other birds under these conditions, which suggests that this compound may play a special role in the pineal physiology of this species. In conclusion, our results show that the daily variations in the metabolism of melatonin-synthesis–related indoles in the goose pineal gland are generated endogenously and controlled by environmental light conditions, as in other birds. However, comparison of the results obtained with the goose to those obtained with other species (chicken, duck) unambiguously shows that the profiles of pineal indoles differ markedly between species, in both the quantitative proportions of the compounds and the characteristics of the diurnal changes. These findings provide strong arguments for the need for comparative studies.     

Effects of duration of daily illumination on reproductive organs and fertility of the meadow vole (Microtus pennsylvanicus).

The objective of this study was to determine the optimum simple, constant photoperiod for voles in a laboratory colony. Voles from an established colony maintained at 22 degrees C with a photoperiod of 14:10 hours of light:dark were transferred at 50 days of age to a photoperiod of 12:12, 14:10, 16:8 or 18:6 hours of light:dark. From Day 96 to Day 102, the 9--10 females per treatment group were paired with a male. Body and gonadal weights, spermatogenesis, ovarian activity and pregnancy were evaluated on Day 110. Reproductive function and body weight of both male and female voles maintained on a photoperiod of 16:8 exceeded (p less than 0.05) values for voles exposed to 12:12 hours of light:dark and tended to be more favorable than for voles in the 14:10 and 18:6 groups.     

Entrainment of circadian rhythms of cholesterol‐LDL,corticosterone and motor activity by feeding schedules in rats

Abstract

The daily variations of locomotor activity, plasma and adrenal corticosterone levels and cholesterol‐LDL were studied in male Wistar rats with food ad libitum and feeding restricted to the first 4 hours of the light phase in LD 12:12..

Under LD 12:12 (light on from 9:00 to 21:00h) rats with food ad libitum were eating and moving during the dark period and the locomotor activity clearly showed a biphasic pattern with three harmonic components. Plasma and adrenal corticosterone levels increased during the light period and reached a maximum value just before the dark period whereas the acrophase of cholesterol‐LDL is found at the beginning of the light phase.

The acrophases of activity, plasma and adrenal corticosterone levels in the restricted feeding schedule rats occurred in the first three hours of lighting and the cholesterol‐LDL acrophase at the beginning of the dark phase.

These results confirm a previous report that the shift of feeding to the light phase seems to cause a concomitant phase‐shift in all the variables measured.     

Serotonin N-acetyltransferase (NAT) induction in mammalian retina: role of cyclic AMP and calcium ions.

In retinas and pineal glands of rat, rabbit and hen, activities of the penultimate (and key regulatory) enzyme in melatonin biosynthesis, serotonin N-acetyltransferase (NAT), display distinct diurnal variations, with high and low values during dark and light phase of a 12-h dark: 12-h light illumination cycle. Two-hour incubation (during daytime hours in light) of isolated pineal glands of the studied vertebrates, or the retinas, with 50 microM forskolin (plus 100 microM 3-isobutyl-1-methylxanthine, IBMX-a phosphodiesterase inhibitor), and 1 mM dibutyryl-cAMP, markedly increased the tissue NAT activity. The same procedures significantly enhanced the enzyme activity of rat retina in light, however, only during nighttime hours. The forskolin (+ IBMX)-induced increase of NAT activity in rat retina was significantly lower in a calcium-free medium, and substantially enhanced when calcium concentration was raised from 1.3 mM to 3.9 mM. Treatment of rats with IBMX or aminophylline, and rabbits with aminophylline, increased NAT activity in their pineal glands irrespective of the time of the day, whereas both phosphodiesterase inhibitors significantly increased the enzyme activity of rat retina only when injected during the subjective dark hours. It is concluded that, by analogy to vertebrate pineal gland, in vertebrate retina an increase of NAT activity (and consequently melatonin formation), stimulated both physiologically (i. e. at night), or pharmacologically, involves a cAMP- and calcium dependent process of the enzyme induction.     

Photoneural Regulation of Rat Pineal Nitric Oxide Synthase

Abstract: We report here a photoneural regulation of nitric oxide synthase (NOS) activity in the rat pineal gland. In the absence of the adrenergic stimulation following constant light exposure (LL) or denervation, pineal NOS activity is markedly reduced. A maximal drop is measured after 8 days in LL. When rats are housed back in normal light-dark (LD) conditions (12:12), pineal NOS activity returns to normal after 4 days. A partial decrease in pineal NOS activity is also observed when rats are placed for 8 days in LD 18:6 or shorter dark phases, indicating that pineal NOS activity reflects the length of the dark phase. Because it is known that norepinephrine (NE) is released at night from the nerve endings in the pineal gland and this release is blocked by exposure to light, our data suggest that NOS is controlled by adrenergic mechanisms. Our observation may also explain the lack of cyclic GMP response to NE observed in animals housed in constant light.     

Progesterone inhibits, on a circadian basis, the release of melatonin by rat pineal perifusion

The effects of 10(-6) and 10(-9) M of progesterone were documented on isoproterenol-stimulated melatonin release by perifused pineal glands removed from female rats in diestrous at two different times of a 12 : 12 h light/dark cycle, 7 and 19 h after light onset (which corresponds to daytime and nighttime, respectively), to look for the existence of a circadian stage-dependence of the hormone effects. Three weeks before the experiment, the rats were synchronized with a 12 : 12 lighting regimen. Progesterone decreased by approximately 50% the release of melatonin during the light span, but not during the dark span. These results show the direct effects of this ovarian hormone on pineal melatonin release and strongly suggest a time-related effect of progesterone on pineal function.     

Darkness-induced changes in noradrenergic input determine the 24 hour variation in beta-adrenergic receptor density in the rat pineal gland: in vivo physiological and pharmacological evidence

In male rats housed under a 14:10 LD cycle (lights on at 0600 h), pineal beta-adrenergic receptors, assessed as 125Iodopindolol (IPIN) binding to membrane preparations, showed a 24 hour variation characterized by a nocturnal increase that peaked around middark (2300 h-0200 h) and a decrease during the latter half of the dark period. Animals exposed to light for 3 hours into the normal dark period showed a similar increase in IPIN binding that was prevented by a single sc injection (0.5 mg/kg) of isoproterenol (ISO). The decrease in IPIN binding observed after middark was prevented both by moving the animals to light at 0200 h and by propranolol administration (20 mg/kg). Likewise, the reduction in IPIN binding was induced in light exposed animals both by ISO administration (in a dose dependent manner) and by injection of norepinephrine (NE) plus the catecholamine uptake blocker desmethylimipramine (DMI). DMI alone was without effect. Chronic denervation of the pineal gland by superior cervical ganglionectomy (SCGx) increased IPIN binding to levels not higher than those observed at middark. The results suggest that rat pineal beta-adrenergic receptors are regulated in a rhythmic 24 hour pattern. A decrease in density (downregulation) induced by a darkness-associated increase in NE release, occurs late in the night before lights on; recovery from the down regulated state (upregulation) occurs during the light and early dark phase, reaching a maximum density of beta-adrenergic receptors at middark not different from that observed in chronically denervated pineal glands.     

Induction of c-fos protein-like immunoreactivity in the rat and hamster pineal gland after the onset of darkness

Summary Induction of c-fos protein (FOS) after the onset of darkness was studied immunocytochemically in the rat and hamster pineal gland. The animals were kept on a 12:12 h light-dark cycle. Before the dark period no FOS staining was seen in either rat or hamster pineal cells. Five hours after the onset of darkness 342±18 pinealocytes/0.2 mm² (mean±SD) displayed FOS-like immunoreactivity in the hamster pineal gland; in the rat pineal gland only 5±2 pinealocytes/0.2 mm² showed a faint staining. Two hours later the density of FOS positive cells was decreased to 60±11/0.2 mm² in the hamster but increased to 519±103/0.2 mm² in the rat pineal gland. Three hours before the beginning of the light period no FOS positive cells were detected in either animal. Both the rat and hamster pineal gland showed a transient and temporally defined expression of c-fos protein in the middle of the dark period. This may be related to a more active functional state of pinealocytes, which is reflected in a peak of melatonin synthesis during the darkness.     

Pineal N-acetyltransferase, pineal and serum melatonin response to isoproterenol stimulation in underfed male rats

Adult male rats were subjected to 4 weeks of 50% food restriction under lighting regimen of 14 h light and 10 h dark. The pineal response to isoproterenol (ISO) was determined. In the time-course study, animals were injected with 0.5 mg/Kg ISO subcutaneously (SC) and killed at different times up to 180 min post injection. In the dose-response study, various doses of ISO (0.2 mg/Kg to 5.0 mg/Kg) were injected intraperitoneally (IP) and animals were killed 120 min post injection. Body weight, pineal N-acetyltransferase (NATase), pineal and serum melatonin (MT) were determined. After 4 weeks of restricted feeding, body weight was reduced by 40%. In the time-course study, peak pineal NATase occurred 120 min post injection in the ad libitum fed animals. By contrast, the food restricted animals showed a gradual increase of pineal NATase up to 180 min post injection. In the dose-response study, the ad libitum fed animals demonstrated a dose dependent increase of pineal NATase up to 5 mg/kg dose. The food restricted animals, however, achieved their maximal pineal NATase at 1 mg/Kg dose with no further increment at 5 mg/Kg dose. These differences in responsiveness were also reflected in pineal and serum MT levels. These results indicate that underfed animals have abnormal pineal NATase, pineal and serum MT responses to ISO stimulation.     

Adrenergic Control of Rat Pineal NO Synthase

Abstract: We have previously shown that exposure of rats to constant light (LL) induced a decrease in NO synthase (NOS) activity in the pineal gland. We present here the evidence that chronic (5 days) norepinephrine (NE) or isoproterenol treatment prevents the effect of LL and enhances pineal NOS activity in LL animals. This effect of NE appears to be mediated by β-adrenoceptors, because it was not mimicked by the α-agonist phenylephrine. Pineal NOS activity was reduced in 16-h light/8-h dark animals treated for 4 days with the β-adrenergic antagonist propranolol but not with the α₁-antagonist prazosin, indicating again an involvement of β-adrenergic receptor in the control of NOS. Treatment with adrenergic antagonists did not affect cortical NOS activity, suggesting that the control of NOS is different in these two tissues or that the pineal expresses a specific isoform of the enzyme. Taken together, these data suggest that NE controls NOS in the pineal gland through β-adrenergic receptors. To our knowledge, this represent the first demonstration of a regulation of NOS by a neurotransmitter in the CNS, as assayed under V _max conditions.