Temporal relationship between melatonin and cortisol responses to nighttime physical stress in humans.

It has been shown that, in the rat, physical stress decreases pineal melatonin levels at night, whereas it increases melatonin production during the day. We have demonstrated that nighttime physical exercise is able to blunt the nocturnal surge of plasma melatonin in healthy subjects. Since this effect might be mediated by exercise-induced cortisol secretion from the adrenal gland, in the present investigation we studied the relationship between cortisol and melatonin responses to nighttime physical stress in six healthy men, aged 28-33 yr. The physical stress consisted of bicycling on a bicycle ergometer at 50% of personal maximum work capacity (MWC), followed by another 10 min of bicycling at 80% of MWC. According to our previous data, physical exercise performed between 2240 h and 2300 h significantly reduced the nocturnal surge of plasma melatonin and increased the levels of cortisol. The surge in plasma cortisol preceded the decrease in plasma melatonin concentration. These findings suggest a temporal relationship between plasma cortisol and melatonin responses to physical stress; the causal nature of this relationship remains to be elucidated.     

Influence of light irradiance on hydroxyindole-O-methyltransferase activity, serotonin-N-acetyltransferase activity, and radioimmunoassayable melatonin levels in the pineal gland of the diurnally active Richardson's ground squirrel

When Richardson's ground squirrels were kept under light:dark cycles of 14:10 h there was no nocturnal rise in pineal hydroxyindole-O-methyltransferase (HIOMT) activity. Conversely, the 10 h dark period was associated with large nocturnal rises in both pineal serotonin-N-acetyltransferase (NAT) activity and radioimmunoassayable melatonin levels. The nighttime rises in pineal NAT and melatonin were not suppressed by the exposure of the animals to a light irradiance of 925 mu W/cm2 during the normal dark period. On the other hand, when the light irradiance was increased to 1850 mu W/cm2 the rise in pineal NAT activity was eliminated while the melatonin rise was greatly reduced. When ground squirrels were acutely exposed to a light irradiance of 1850 mu W/cm2 for 30 min beginning at 5.5 h after lights out, pineal NAT activity and melatonin levels were reduced to daytime values within 30 min. The half-time (t 1/2) for each constituent was less than 10 min. Exposure to a light irradiance of either 5 s or 5 min (beginning at 5.5 h into dark period) was equally as effective as 30 min light exposure in inhibiting pineal NAT activity and melatonin levels. When animals were returned to darkness after a 30 min exposure to a light irradiance of 1850 mu W/cm2 at night, both pineal NAT activity and melatonin levels were restored to high nighttime levels within 2 h of their return to darkness. The results indicate that the pineal gland of the wild-captured, diurnal Richardson's ground squirrel is 9000 X less sensitive to light at night than is the pineal gland of the laboratory raised, nocturnal Syrian hamster.     

Does bright light suppress nocturnal melatonin secretion more in women than men?

Summary Sex differences in the sensitivity of the human pineal gland to the suppressant effect of bright light on melatonin synthesis were studied in 6 healthy men and women. Blood samples were collected in two randomly ordered sessions: in one, subjects rested supine in bed, in the dark, from 21.00 to 7.00h; in the other session, they were exposed to bright light (2,000 lux) from 2.00 to 4.00 h. In the dark condition, no significant differences were observed between men and women in either the timing or the absolute values of melatonin plasma levels, whereas after bright light exposure, the suppression of plasma melatonin was a 40% greater in women than in men. These findings suggest that, in humans, there is a sex difference in the nocturnal sensitivity of the pineal to light.     

Studies on pineal melatonin levels in a diurnal species,the Eastern chipmunk (Tamias striatus): Effects of light at night,propranolol administration or superior cervical ganglionectomy

Summary Five experiments were carried out on the control of melatonin levels in the pineal gland of a diurnal species, the Eastern chipmunk (Tamias striatus). We confirmed that the exposure of chipmunks to fluorescent white light of 3,981–4,304 lux during the normal dark period does not prevent the rise in pineal melatonin levels normally associated with darkness. Also, the administration of propranolol (20mg/kg) at 8 p.m. did not block the rise in pineal melatonin in animals exposed to either dark or light at night. Similarly, if chipmunks received propranolol 4 hours into the dark phase, pineal melatonin levels were not depressed 2 hours later. When animals were superior cervical ganglionectomized, however, the pineal content of melatonin remained low regardless of whether the animals were exposed to darkness or light at night. The exposure of chipmunks acutely to light at midnight (4 hours after darkness onset) had only a slight depressive effect on pineal melatonin 30 min later; by comparison, when chipmunks were acutely exposed to light at 3 a.m. (7 hours after darkness onset) daytime pineal melatonin levels were reached within 15 min after light onset. These findings in a diurnal species, the Eastern chipmunk, differ markedly when compared to previously reported observations on nocturnal laboratory rodents.     

Changes in Pineal Indoleamines in Rats after Single Melatonin Injections: Evidence for a Diurnal Sensitivity to Melatonin

We recently determined that melatonin stimulated serotonin (5-HT) secretion from rat pineal glands by increasing 5-HT release from the pinealocytes (μM melatonin concentrations) and by inhibiting 5-HT uptake in the pineal sympathetic nerve endings (mM melatonin concentrations). The present study investigated whether a single melatonin injection could alter the content of indoleamines in the rat pineal gland, as well as its possible dependence on the daytime of administration. Melatonin (150 μg/kg) was i.p. injected at 8 time points (11.00 h, 14.00 h, 17.00 h, 20.00 h, 23.00 h, 02.00 h, 05.00 h and 08.00 h) to rats kept in 12:12 h light:dark cycle (lights on at 07.00 h). Melatonin injections in the afternoon (17:00 h) and late in the nighttime (02.00 h and 05.00 h) decreased pineal 5-HT content 90 min later. The levels of 5-hydroxyindoleacetic acid (5-HIAA) were also decreased 90 min after the melatonin treatment at 14.00 h, 17.00 h and 02.00 h. The effect of melatonin on 5-HT content was a long-lasting effect (still evident after 180 min) only when injected at 02.00 h, whereas 5-HIAA levels were found to be decreased 180 min after melatonin treatment at 14.00 h and 23.00 h. No changes in these compounds were detected 240 min after melatonin treatment. Moreover, melatonin did not change 5-hydroxytryptophan levels at any of the daytime points studied. By contrast, 90 min after the injection of melatonin at 20.00 h, an increased content of pineal N-acetylserotonin was observed. This effect of melatonin could be mediated through a phase alteration of the pineal N-acetyltransferase activity rhythm by acting on the suprachiasmatic clock, althought a direct melatonin effect on the pineal rhythmic function cannot be excluded. The effects of the hormone on 5-HT and 5-HIAA contents agree with previous findings on the inhibitory effect of pharmacological doses of melatonin on pineal 5-HT uptake, which presumably would result in a decreased intraneuronal content of 5-HT and its acid metabolite. These data point to an acute regulatory action of exogenous melatonin on the pineal melatonin synthesis pathway which seems to be limited to two daytime phases: the afternoon-early evening period and the second half of the night.     

Effect of dark exposure in the middle of the day on Period1, Period2, and arylalkylamine N-acetyltransferase mRNA levels in the rat suprachiasmatic nucleus and pineal gland

The suprachiasmatic nucleus (SCN) of the mammalian hypothalamus contains a central circadian pacemaker, which adjusts circadian rhythms within the body to environmental light-dark cycles. It has been shown that dark exposure in the day causes phase shifts in circadian rhythms, but it does not induce changes in the melatonin levels in the pineal gland. In this study, we examined the effect of dark exposure on two "circadian clock" genes Period1 and Period2 mRNA levels in the rat SCN, and on Period1, Period2, and arylalkylamine N-acetyltransferase (Aa-Nat, the rate-limiting enzyme in melatonin synthesis) gene expression in the pineal gland. Period1 and Period2 mRNA levels were significantly decreased in the SCN after 0.5 and 2 h, respectively, therefore suggesting that changes in those mRNA levels may be the part of the mechanisms of dark-induced phase shifts. Period1 and Aa-Nat mRNA levels in the pineal gland were not affected by darkness, but Period2 was moderately affected. Since Period1 and Aa-Nat mRNA levels in the pineal gland did not respond to dark stimulation, we further examined whether the pineal gland itself is capable of responding to adrenergic stimulation at this time of the day. Isoproterenol significantly induced Period1 and Aa-Nat mRNA levels; however, it did not affect Period2. Although previous studies have reported that during the day the SCN "gates" the dark information reaching the pineal, our data demonstrate that dark information may reach the pineal during the daytime.     

Neither the pituitary gland nor the sympathetic nervous system is responsible for eliciting the large drop in elevated rat pineal melatonin levels due to swimming

Summary Since the pineal gland is an end organ of the sympathetic nervous system, stress might increase the synthesis of its hormone, melatonin. The stress of a 10 min swim, which elicits a marked rise in circulating catecholamines, causes a dramatic depression of high pineal melatonin levels at night within 15 min after swimming onset. N-acetyltransferase (NAT) activity is unaffected by the treatment at 15 or 30 min after swimming onset. Within 90 min after initiation of a 15 min swim, high nighttime pineal melatonin levels are restored while NAT values remain elevated. The swimming-induced reduction in high pineal melatonin levels is not influenced by either hypophysectomy, superior cervical ganglionectomy, prazosin (₁-adrenergic receptor blocker) pretreatment, yohimbine (₂-adrenergic receptor blocker) pretreatment, or reserpine (amine depletor) pretreatment. These results indicate that neither hormones secreted from the pituitary gland nor catecholamines secreted from the sympathetic nerves are involved in eliciting the dramatic reduction in elevated pineal melatonin levels in the rat.     

Treatment with forskolin for 8 hours during the day increases melatonin synthesis in the Syrian hamster pineal gland in organ culture: the long lag period is required for RNA synthesis.

The exposure of organ-cultured pineal glands of Syrian hamsters to forskolin, an adenylate cyclase activator, caused marked increases in melatonin levels when glands were collected in the second half of the dark period and incubated for 4 h. However, when glands were collected at the beginning of the dark period and incubated with the same drug, a significant increase in melatonin content was observed only after 6-8 h. Likewise, when glands were collected at the beginning of the light period, forskolin stimulated melatonin synthesis only after 6-8 h of incubation with the drug. These results support the existence of a relatively long lag period necessary for the induction of melatonin production in the Syrian hamster pineal gland. Experiments with actinomycin D indicate that RNA synthesis occurs during the lag period; thus, actinomycin D blocks the induction of melatonin synthesis by forskolin in glands collected at the beginning of the dark period and incubated for 8 h. In contrast, when pineal glands were collected from hamsters killed in the second half of the dark period, and incubated with forskolin for either 4 or 8 h, actinomycin D was unable to block the induction of melatonin production. These data suggest that RNA, presumably messenger RNA, which is necessary for increasing hamster pineal melatonin synthesis, is synthesized and accumulates during the first half of the night.     

Response of pineal serotonin N-acetyltransferase activity in male guinea pigs exposed to light pulses at night

Summary Serotonin N-acetyltransferase (NAT), which is crucial for the formation of melatonin, undergoes a typical day/night rhythm in the pineal gland with low levels during daytime and high levels at night. Short pulses of light given at night have been shown to rapidly depress NAT activity in some species, but not in others, the reasons for this difference being unclear. As diurnality and nocturnality of the experimental animals may play a role and since diurnally active animals have been little investigated in this respect, in the present study the diurnally active guinea pig was investigated. Male guinea pigs kept under a lighting regimen of LD 1212 (lights off at 1700 hrs) were killed between 1200 or 1300 hrs and between 0000 and 0200 hrs, at night in the dark or after exposure to 10 or 45 min of light. The results obtained show that the day/night difference of NAT activity is about 2-fold. 10 min or 45 min of light given at night significantly depress pineal NAT activity. Re-exposure to darkness for 1 hr of animals previously given light for 10 min leads to restoration of NAT activity. These findings together with data from the literature suggest that it does not appear to be the activity pattern (diurnality versus nocturnality) of an animal nor the amplitude of the day/night difference of pineal NAT activity that account for the suppressibility or non-suppressibility of pineal NAT activity by light at night.     

Pineal Microdialysis of the Melatonin in Conscious Sheep: Methodology, Application to a Diurnal Rhythm and Effect of Isoproterenol

This paper describes the development of a new technique to measure melatonin contents in the pineal gland of moving sheep: the microdialysis. A dialysis probe was used to collect extracellular fluid in the sheep pineal gland, but also to inject directly into it different drugs such as isoproterenol at a very low concentration. The probe was implanted the day before the beginning of the experiment in order to obtain low levels of melatonin. This technique makes it possible to measure melatonin in the dialysate and plasma of rams submitted to 8L:16D. No melatonin either in the dialysate or in the plasma was found during the light phase. Shortly after lighting off, the melatonin concentration increased in the dialysate and plasma and remained stable during the dark phase. Melatonin concentrations began to decrease before lighting on and no detectable levels were found during the following light phase. The secretion of melatonin is, at least, under adrenergic regulation. Local infusion of isoproterenol (90  μl at 10⁻⁶ M), an agonist of β adrenergic receptor, through the probe, increased melatonin levels during 2  h, even when infusions were repeated 3 times. This demonstrates the presence of β adrenergic receptors. The technique presented in this paper could be of considerable interest for studying sheep pineal gland and its main secretion, melatonin, for example during diurnal rhythms or for studying its regulation.     

Diurnal variation in glucose utilization in the pineal body of the monkey

We employed the quantitative 2-[14C]-deoxyglucose method (Sokoloff's method) to measure glucose utilization in the pineal gland of pubescent monkeys. Glucose utilization in the pineal was 80-110% higher in the nocturnal, awake animals compared to the rate of both groups studied in the nocturnal, awake animals with both eyes open and with light deprivation for three hours. Short term visual deprivation during the day was without effect on pineal glucose utilization. The diurnal variations in melatonin levels in blood and CSF, higher at night than during the day, are the result of corresponding changes in the rate of production and elaboration of melatonin in the pineal gland. The release of norepinephrine from the postganglionic fiber of the superior cervical ganglia controls the production of melatonin in the pineal by regulating the activity of serotonin-N-acetyltransferase. It was reported that electrical stimulation of SCG via sympathetic trunk increased the levels of serotonin-N-acetyltransferase in the pineal and that it also increased glucose utilization in the pineal. It is believed that metabolic increase in the pineal reflects increased activity in sympathetic terminals distributed throughout the gland which stimulate its increase in hormone production. The present results indicate that there is an elevation of pineal metabolic rate at a time when blood and CSF levels of melatonin are known to be elevated Our finding that short-term light deprivation during the day did not affect the pineal metabolic rate is consistent with the result by Reppert et al (1981) in which they found that exposure to darkness during the day does not result in an increase in CSF melatonin.     

The depression in rat pineal melatonin production after saline injection at night may be elicited by corticosterone

A hind-leg subcutaneous saline injection into rats at night elicits a decrease in N-acetyltransferase (NAT) activity and melatonin content of the pineal gland. The decrement in pineal melatonin production after saline injection is prevented by adrenalectomy. The present studies were undertaken to determine what factor(s) from the adrenal gland cause(s) the drop in pineal melatonin production after saline injection at night. In the first study, groups of intact and adrenal-demedullated male rats were given a saline injection at 23.10 h (3 h, 10 min after lights off) and their pineals were collected 15 or 30 min later. Pineal NAT activity was depressed in both intact and adrenal-demedullated rats at 15 min postinjection as compared to their respective control animals. Pineal melatonin levels exhibited a drop in intact animals at 15 min and in adrenal-demedullated rats at 30 min. In a second study, hypophysectomy was found to prevent the drop in nocturnal pineal NAT activity and melatonin levels normally associated with a hind leg injection of saline. Finally, in a third experiment, groups of hypophysectomized rats were injected i.p. with corticosterone at 23.10 h and killed 10, 25 or 40 min postinjection. Corticosterone injection in hypophysectomized rats produced a response similar to that caused by saline injection in intact animals: NAT activity was depressed at 10 min and melatonin content was lowered at 25 min. These results suggest that the adrenal-mediated depression in melatonin synthesis after saline injection at night in rats may be elicited by an adrenal cortical hormone (corticosterone) and apparently does not involve the release of factors from the adrenal medulla.     

Effect of ageing on melatonin synthesis induced by 5-hydroxytryptophan and constant light in rats.

1. This paper describes the effect of the serotonin precursor 5-hydroxytryptophan (5-HTP) on pineal melatonin synthesis young and old rats. 2. 5-HTP itself increased pineal melatonin levels in old rats but did not change melatonin concentrations in young rats kept under 12 hr/12 hr light/dark conditions. 3. After continuous exposure to light for 72 hrs, 5-HTP induced a significant increase in melatonin levels in young rats but did not change the 5-HTP effect on melatonin in old animals. 4. These results are discussed in consideration of previous reports of altered beta-receptor up-regulation by light in old animals, and suggest that the age-related decrease in melatonin synthesis is not entirely related to changes of the enzymatic machine for melatonin synthesis.     

Increased pineal melatonin content coupled to restricted water availability in a Pavlovian conditioning paradigm in rats

Summary The objective of this study was to assess whether rat pineal melatonin content could be modified in a classical conditioning paradigm. In rats kept under light (200 lux) from 06.00 to 18.00h daily, the time of lights off was selected as the unconditioned stimulus (US). Restricted water availability (from 10 min before to 10 min after light-dark, LD, transition) was the conditioned stimulus (CS). The conditioned and unconditioned responses were measured as the changes in pineal melatonin levels 4 h after LD transition. In animals under regular lighting conditions, lights out at 18.00 h (the US) caused a 4.4–7.8-fold increase of pineal melatonin concentration 4 h after later, when compared to animals maintained under light for the 4 h-period. After a training period of 7 days of restricted water availability (the CS), significantly augmented pineal melatonin levels were found in rats that were exposed to water but were maintained under light for the 4 h period after expected LD transition. The control animals for this experiment, i.e., rats which had undergone the training period, were kept for 4 h under light after expected LD transition, and did not receive water at LD transition, exhibited very low pineal melatonin levels. The conditioned increase of pineal melatonin content attained lower values than those in rats exposed to normal lighting conditions. It also fulfilled the contingency criterion, that is, it caused at trial a significant elevation of pineal melatonin content only when water availability was applied from 10 min previously to LD transition during training, and not 20 min after LD transition. After a training period of 7 days, restricted water availability applied 4 h before lights off (at 14.00 h), caused an enhanced production of melatonin 4 h later, regardless of the animals being exposed either to a dark or to a light environment. The results indicate that pineal melatonin production can be manipulated in a classical conditioning paradigm, when an appropriate CS stimulus is used.     

Circadian rhythm of melatonin release in pineal gland culture: arg-vasopressin inhibits melatonin release

The mammalian pineal gland is known to receive a noradrenergic sympathetic efferent signal from the suprachiasmatic nucleus (SCN) via the superior cervical ganglion. Arg-vasopressin (AVP) containing neurons in the SCN is one of the output paths of circadian information to the other brain areas. AVP release from the SCN is suppressed by melatonin. In turn, we determined the direct effect of AVP on melatonin release using pineal gland explant culture. AVP (1 μM) suppressed melatonin release. Noradrenaline stimulated melatonin release was attenuated by AVP. In turn, the expression of the melatonin synthesis enzyme arylalkylamine N-acetyltransferase mRNA in the rat SCN was reported. We measured melatonin content in the SCN in rats kept under the light–dark cycle and constant dim light. Melatonin in the SCN was higher during the dark period than that in the light. A similar tendency was also observed in the SCN of animals kept under a constant dim light. It was suggested that the reciprocal regulation of melatonin release and AVP release occurs in the SCN and pineal gland.     

In vivo and in vitro studies on modulation of the pineal endocrine function by L-acetyl-carnitine in the rat.

Male Sprague-Dawley rats injected (i.p.) at 1500h with L-acetyl-carnitine in doses of 10, 30 or 90 mg/kg exhibited a notable increase in their pineal and serum melatonin content 1 hr later. Likewise, L-acetyl-carnitine administered in the same dose range induced a significant increase of pineal and serum melatonin content in rats treated at 0100h, following exposure of 30 min to bright white light to suppress endogenous melatonin. Under in vitro experimental conditions, however, 60 min of coincubation of isolated rat pineal glands with L-acetyl-carnitine (10(-5) M) did not result in an elevation in melatonin accumulated in the incubation medium. These results demonstrate that, in vivo, L-acetyl-carnitine can exert a modulatory action on synthesis and release of melatonin, possibly by modifying noradrenergic transmission and signal transduction in the pineal gland.     

Tetrodotoxin administration in the suprachiasmatic nucleus prevents NMDA-induced reductions in pineal melatonin without influencing Per1 and Per2 mRNA levels

The suprachiasmatic nucleus (SCN) of the anterior hypothalamus contains a light-entrainable circadian pacemaker. Neurons in the SCN are part of a circuit that conveys light information from retinal efferents to the pineal gland. Light presented during the night acutely increases mRNA levels of the circadian clock genes Per1 and Per2 in the SCN, and acutely suppresses melatonin levels in the pineal gland. The present study investigated whether the ability of light to increase Per1 and Per2 mRNA levels and suppress pineal melatonin levels requires sodium-dependent action potentials in the SCN. Per1 and Per2 mRNA levels in the SCN and pineal melatonin levels were measured in Syrian hamsters injected with tetrodotoxin (TTX) prior to light exposure or injection of N-methyl-D-aspartate (NMDA). TTX inhibited the ability of light to increase Per1 and Per2 mRNA levels and suppress pineal melatonin levels. TTX did not, however, influence the ability of NMDA to increase Per1 and Per2 mRNA levels, though it did inhibit the ability of NMDA to suppress pineal melatonin levels. These results demonstrate that action potentials in the SCN are not necessary for NMDA receptor activation to increase Per1 and Per2 mRNA levels, but are necessary for NMDA receptor activation to decrease pineal melatonin levels. Taken together, these data support the hypothesis that the mechanism through which light information is conveyed to the pacemaker in the SCN is separate from and independent of the mechanism through which light information is conveyed to the SCN cells whose efferents suppress pineal melatonin levels.     

Effect of different photoperiods on the diurnal rhythm of 5-methoxytryptamine in the pineal gland of golden hamsters (Mesocricetus auratus)

Summary This study tested the photo-dependency of the rhythmic synthesis of 5-methoxytryptamine (5-MT) in the pineal gland of golden hamsters. After pargyline administration, pineal 5-methoxytryptamine and melatonin were measured by HPLC in male golden hamsters kept under short and long photoperiod. In both photoperiodic regimes, a clear 5-MT rhythm was observed which fitted a sinusoidal function with high values occuring during the day-time and low values occuring during the night-time. The duration of the low nighttime levels was clearly proportional to the length of the dark phase. A marked rhythm of melatonin synthesis was also seen with low daytime levels and high night-time values. An inverse relationship between 5-MT and melatonin levels was observed. Thus, after pargyline administration, the rhythms of 5-MT and melatonin in the pineal gland of golden hamsters are photoperiod-dependent and show a reciprocal relationship.     

Effects of thyroid hormone on light/dark melatonin synthesis and release by young and maturing rat pineal glands in vitro

Synthesis and release of melatonin were studied in pineal explants from 14- (young) and 60-day-old ('maturing') male Long-Evans rats with or without added thyroid hormone, triiodothyronine (T3), at or near physiological levels and under light and dark conditions. Incubation for 6 hr (1200-1800) was in a synthetic medium; melatonin was measured by radioimmunoassay (RIA). In light, T3 increased melatonin levels in pineal and medium of cultures from either young or maturing animals. In dark, T3 decreased melatonin levels in the pineals of either age, but was without significant effect on levels in the medium. Since it is known from other work that 14-day-old rat pineal glands do not yet have a complete sympathetic innervation system, it is here doubly evident that T3 can modulate directly pineal synthesis and release of melatonin, and may not depend upon a mature sympathetic innervation. Light in the studied conditions was permissive from the stimulatory action of T3 on pineal synthesis and release of melatonin in vitro.