The human pineal gland and melatonin in aging and Alzheimer's disease

The pineal gland is a central structure in the circadian system which produces melatonin under the control of the central clock, the suprachiasmatic nucleus (SCN). The SCN and the output of the pineal gland, i.e. melatonin, are synchronized to the 24-hr day by environmental light, received by the retina and transmitted to the SCN via the retinohypothalamic tract. Melatonin not only plays an important role in the regulation of circadian rhythms, but also acts as antioxidant and neuroprotector that may be of importance in aging and Alzheimer's disease (AD). Circadian disorders, such as sleep-wake cycle disturbances, are associated with aging, and even more pronounced in AD. Many studies have reported disrupted melatonin production and rhythms in aging and in AD that, as we showed, are taking place as early as in the very first preclinical AD stages (neuropathological Braak stage I-II). Degeneration of the retina-SCN-pineal axis may underlie these changes. Our recent studies indicate that a dysfunction of the sympathetic regulation of pineal melatonin synthesis by the SCN is responsible for melatonin changes during the early AD stages. Reactivation of the circadian system (retina-SCN-pineal pathway) by means of light therapy and melatonin supplementation, to restore the circadian rhythm and to relieve the clinical circadian disturbances, has shown promising positive results.     

Nervous mechanisms of restraint water-immersion stress-induced gastric mucosal lesion

Stress-induced gastric mucosal lesion(SGML) is one of the most common visceral complications after trauma. Exploring the nervous mechanisms of SGML has become a research hotspot. Restraint water-immersion stress(RWIS) can induce GML and has been widely used to elucidate the nervous mechanisms of SGML. It is believed that RWIS-induced GML is mainly caused by the enhanced activity of vagal parasympathetic nerves. Many central nuclei, such as the dorsal motor nucleus of the vagus, nucleus of the solitary tract, supraoptic nucleus and paraventricular nucleus of the hypothalamus, mediodorsal nucleus of the thalamus, central nucleus of the amygdala and medial prefrontal cortex, are involved in the formation of SGML in varying degrees.Neurotransmitters/neuromodulators, such as nitric oxide, hydrogen sulfide,vasoactive intestinal peptide, calcitonin gene-related peptide, substance P,enkephalin, 5-hydroxytryptamine, acetylcholine, catecholamine, glutamate, γ-aminobutyric acid, oxytocin and arginine vasopressin, can participate in the regulation of stress. However, inconsistent and even contradictory results have been obtained regarding the actual roles of each nucleus in the nervous mechanism of RWIS-induced GML, such as the involvement of different nuclei with the time of RWIS, the different levels of involvement of the sub-regions of the same nucleus, and the diverse signalling molecules, remain to be further elucidated.     

Phase-shifting effects of light on the circadian rhythms of 5-methoxytryptophol and melatonin in the chick pineal gland

In the chick pineal gland, 5-methoxytryptophol and melatonin concentrations fluctuate in a rhythmic manner. These rhythms are circadian in nature persisting in constant darkness and have opposite phases. Acute exposure of chicks to white light (30 lux for 5, 10, 20, and 30 min) at night increased the amount of pineal 5-methoxytryptophol and decreased pineal melatonin content. A 6 hr pulse of light (100 lux) applied early in the subjective night (CT12-CT18) caused a delay in the phase of the circadian rhythms of 5-methoxytryptophol and melatonin by 3.7 and 4.5 h, respectively, compared to untreated controls. When the 6 hr light pulse was given during the late subjective night (C18 CT24) it advanced the phase of the 5-methoxytryptophol and melatonin rhythms by 8.1 and 11.9 h, respectively. In the chick pineal the phase-advancing effects of light on the circadian rhythms of 5-methoxytryptophol and melatonin were more pronounced than the phase-delaying effects. Our results provide the first evidence that light is capable of phase shifting the 5-methoxytryptophol rhythm in a manner similar to its action on the melatonin rhythm.     

Posthatching developmental changes in noradrenaline content in the chicken pineal gland

Noradrenaline (NA) levels in pineal gland of chickens at various posthatching stages (P2, P4, P8, P15, P30 and P57) were determined by high-performance liquid chromatography with electrochemical detection. Pineal NA content markedly increased between P2 and P30. P30 and P57 chickens, kept from the day of hatching under a 12:12 hr light-dark (LD) illumination cycle, exhibited rhythmic changes in pineal NA, with levels in the dark period being markedly higher than in the light period. In younger birds pineal NA concentrations did not show pronounced daily variations. In 4-wk-old chickens (P28-30) kept under constant darkness (DD), the rhythmic pattern of pineal NA persisted for 1 day (with higher values during the subjective dark phase than during the subjective light phase), but this disappeared 24 hr after the introduction of DD. In contrast, NA content in pineal glands isolated from birds maintained for 2 days under continuous light was similar to that found during the light phase of the LD cycle, and did not exhibit significant rhythmicity. In P30 chickens, pretreated with alpha-methyl-p-tyrosine (AMPT, an inhibitor of tyrosine hydroxylase, the key regulatory enzyme in the biosynthesis of catecholamines), pineal NA content declined slowly and monophasically during the light phase. During the dark phase the AMPT-induced decay of NA was biphasic--namely an initial rapid decline over the first 15 min which was followed by a slow-rate decline--an observation indicating that NA turnover was higher in the dark. Acute exposure of the dark-adapted P30 and P57 chickens to light significantly decreased pineal NA content, but did not affect pineal NA concentrations in younger birds. Our results suggest that the NA rhythm in the chicken pineal gland and its sensitivity to light regulation progressively develop during the first month of life.     

Estrogen modulates the nocturnal synthesis of melatonin in peripubertal female rats

Abstract: Our objective was to evaluate the effects of estrogen deficit and of estrogen stimulation on the synthesis of pineal melatonin in female rats during the peripubertal period. The levels of melatonin and N-acetylserotonin (NAS) and the activities of N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) were determined in homogenates of pineal glands obtained from peripubertal female Sprague- Dawley rats 4 to 12 weeks of age in the mid-dark during the daily light/dark cycle. Animals were ovariectomized at 4 weeks of age; daily administration of estradiol benzoate (E₂B, 1.0 μg/d, s.c.) was initiated at 4 weeks of age. A peak in the pineal levels of melatonin and NAS and in NAT activity was observed in untreated (control) rats with intact ovaries at 6 weeks. HIOMT activity increased from Week 4 to 6 and remained unchanged thereafter. Ovariectomy at Week 4 led to significant increases in the levels of melatonin and of NAS and NAT in activity at Week 8. NAT activity Week 10 resembled that of control animals, but levels of melatonin and NAS were slightly elevated. Ovariectomy did not affect HIOMT activity. Subcutaneous injection of E₂B significantly decreased the levels of melatonin and NAS and of NAT activity at Week 4, as compared with those in control rats. E₂B suppressed the ovariectomy-induced elevation of levels of melatonin and NAS and of NAT activity, similar to the effect in control animals. E₂B did not affect HIOMT activity. Our results suggest that estrogen modulates the nocturnal synthesis of melatonin in the pineal gland in peripubertal female rats. The effects of estrogen on melatonin synthesis appeared to be mediated by the modulation of NAT activity.     

Evidence for differential photic regulation of pineal melatonin synthesis in teleosts

The aim of this study was to compare the circadian control of melatonin production in teleosts. To do so, the effects of ophthalmectomy on circulating melatonin rhythms were studied along with ex vivo pineal culture in six different teleosts. Results strongly suggested that the circadian control of melatonin production could have dramatically changed with at least three different systems being present in teleosts when one considers the photic regulation of pineal melatonin production. First, salmonids presented a decentralized system in which the pineal gland responds directly to light independently of the eyes. Then, in seabass and cod both the eyes and the pineal gland are required to sustain full night-time melatonin production. Finally, a third type of circadian control of melatonin production is proposed in tilapia and catfish in which the pineal gland would not be light sensitive (or only slightly) and required the eyes to perceive light and inhibit melatonin synthesis. Further studies (anatomical, ultrastructural, retinal projections) are needed to confirm these results. Ex vivo experiments indirectly confirmed these results, as while the pineal gland responded normally to day-night rhythms in salmonids, seabass and cod, only very low levels were obtained at night in tilapia and no melatonin could be measured from isolated pineal glands in catfish. Together, these findings suggest that mechanisms involved in the perception of light and the transduction of this signal through the circadian axis has changed in teleosts possibly as a reflection of the photic environment in which they have evolved in.     

Effects of cyclosporin A on water-immersion stress-induced gastric lesion and gastric secretion in rats

Cyclosporin A is an immunosuppressive agent which is well known as a specific inhibitor of calcineurin (protein phosphatase 2B). In this study, we investigated the effects of cyclosporin A on water-immersion stress-induced gastric ulcer formation and gastric acid secretion in rats. We also examined the localization of calcineurin immunohistochemically. Calcineurin was specifically expressed in gastric parietal cells and chief cells of the gastric mucosa. The intraperitoneal administration of cyclosporin A dose-dependently suppressed the development of gastric mucosal lesions induced by water-immersion stress and inhibited gastric acid secretion, as assessed by pylorus ligation. These results indicated that calcineurin may play an important role in gastric acid secretion. Received: October 14, 1999 / Accepted: January 28, 2000     

HPLC validation of a circadian melatonin rhythm in the pineal gland of inbred mice

Abstract: Production of melatonin in the pineal gland of inbred mice such as C57B1/6J, B ALB/c and AKR strains is still a matter of debate. In previous studies, we and other authors showed that these strains of inbred mice have a clear-cut circadian rhythm of serum melatonin and urinary 6-hydroxy-melatonin-sulphate. In contrast, other groups claimed these mice are unable to synthesize melatonin. These studies were based on RIA measurements and/or estimates of N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) activities. In the present study we validate the presence of melatonin in the pineal gland of C57B1/6, BALB/c, and AKR mice by HPLC determinations. We found a short-term melatonin peak in the middle of the dark period with a pattern which mirrors that found previously in the serum. The possibility remains, although it seems unlikely, that the pineal melatonin rhythm measured here represents melatonin produced elsewhere which then was subsequently taken up by the pineal gland.     

Daily variation in the concentration of 5-methoxytryptophol and melatonin in the duck pineal gland and plasma

The duck pineal gland rhythmically produces two 5-methoxyindole compounds, i.e. 5-methoxytryptophol and melatonin. 5-Methoxytryptophol levels are low at night and high during the day, while melatonin concentrations are high at night and low during the day. The melatonin rhythm reflects oscillations in the activity of serotonin N-acetyltransferase (AA-NAT; a penultimate and key regulatory enzyme in the melatonin biosynthetic pathway). The activity of hydroxyindole-O-methyltransferase (HIOMT; an enzyme involved in the synthesis of both 5-methoxytryptophol and melatonin) does not exhibit any significant rhythmic changes throughout the 24-hr period. Plasma levels of melatonin exhibited daily changes that were parallel to fluctuations in pineal melatonin content. Although plasma concentrations of 5-methoxytryptophol were low in ducks, they showed daily variations. The mean 5-methoxytryptophol concentration between zeitgeber time 9 (ZT9) and ZT15 was 2.4-times higher than the mean value for samples collected between ZT18 and ZT3. These findings indicate that in the duck the pineal production of 5-methoxytryptophol and melatonin may be inversely correlated.     

No difference in day-night serum melatonin concentration after pineal grafting into the third cerebral ventricle of pinealectomized rats

Serum melatonin concentration and its day-night difference in pinealectomized, stereotaxically grafted rats with pineal transplants was examined. The nighttime serum melatonin concentration increased significantly only in pinealectomized rats that received two pineal transplants. In neither pinealectomized rats receiving two pineal glands and one cotransplant of superior cervical sympathetic ganglion nor pinealectomized-ganglionectomized rats receiving two pineal transplants was there a demonstrable increased in serum melatonin concentration. Although some pineal transplanted hosts demonstrated increases in serum melatonin concentration, there was no day-night variation in serum melatonin concentration. This may be due to a number of mitigating factors. Pineal grafts may not receive the appropriate functional reinnervation from the host brain due to the location of the transplantation in the cerebral ventricle or due to lack of sufficient time for the growth of invading host neurites.     

Interruption of nocturnal pineal melatonin synthesis in spontaneous recrudescent Djungarian hamsters (Phodopus sungorus)

The duration of nighttime synthesis of the pineal hormone melatonin is believed to determine the breeding season in many mammalian species. Hamsters exposed to short days undergo gonadal involution followed by a return to normal function, suggesting a developed insensitivity to regressive photoperiods. This recrudescence may be due to either exhaustion of the pineal or to target-desensitization. Both theories have been tested previously but failed to explain this phenomenon. We performed an experiment in the Djungarian hamster (Phodopus sungorus), a well-characterized photosensitive species with a type C melatonin pattern (prolonged peak during majority of dark phase), in an attempt to resolve this issue. Among age-matched male hamsters exposed to short days for either 16 weeks (involuted) or 38 weeks (spontaneous recrudescent), marked phase differences in diurnal pineal melatonin rhythms were observed. Furthermore, in recrudescent hamsters the melatonin pattern was divided into two parts, possibly no longer recognizable as a typical short-day rhythm.     

Perinatal development of circadian melatonin production in domestic chicks

In contrast to the situation in mammals, in which circadian melatonin production by the pineal gland does not begin until some time after birth, the development of pineal gland rhythmicity is an embryonic event in the precocial domestic fowl. A distinct melatonin rhythm was found in 19-d-old chick embryos maintained under light:dark (LD) 16:8. No significant variation in melatonin levels was detected in embryos exposed to LD 8:16. The melatonin rhythm in the pineal gland and plasma of chick embryos incubated for 18 d in LD 12:12 persisted for 2 d in constant darkness indicating that melatonin production is under circadian control at least from the end of embryonic life. A 1-d exposure to a LD cycle during the first postembryonic day was sufficient to entrain the melatonin rhythm, and previous embryonic exposure to either LD or constant darkness (DD) neither modified this rapid synchronization nor did it affect the melatonin pattern during the two subsequent days in DD. It is suggested that, in contrast to the situation in mammals, the avian embryo has evolved its own early circadian melatonin-producing system because, as a consequence of its extrauterine development, it cannot use the system of its mother.     

Serotonin modulates melatonin synthesis as an autocrine neurotransmitter in the pineal gland

The pineal gland secretes melatonin principally at night. Regulated by norepinephrine released from sympathetic nerve terminals, adrenergic receptors on pinealocytes activate aralkylamine N-acetyltransferase that converts 5-hydroxytryptamine (5-HT, serotonin) to N-acetylserotonin, the precursor of melatonin. Previous studies from our group and others reveal significant constitutive secretion of 5-HT from pinealocytes. Here, using mass spectrometry, we demonstrated that the 5-HT is secreted primarily via a decynium-22–sensitive equilibrative plasma membrane monoamine transporter instead of by typical exocytotic quantal secretion. Activation of the endogenous 5-HT receptors on pinealocytes evoked an intracellular Ca²⁺ rise that was blocked by RS-102221, an antagonist of 5-HT_2C receptors. Applied 5-HT did not evoke melatonin secretion by itself, but it did potentiate melatonin secretion evoked by submaximal norepinephrine. In addition, RS-102221 reduced the norepinephrine-induced melatonin secretion in strips of pineal gland, even when no exogenous 5-HT was added, suggesting that the 5-HT that is constitutively released from pinealocytes accumulates enough in the tissue to act as an autocrine feedback signal sensitizing melatonin release.

The principal role of pinealocytes of the pineal gland (PG) is to synthesize and secrete the hormone melatonin, driven by the neurotransmitter norepinephrine (NE) released at night from sympathetic nerve terminals. Acting through α₁- and β₁-adrenergic receptors on pinealocytes, the adrenergic signal generates Ca²⁺ and cyclic AMP (cAMP) to activate up to 150-fold (in rat) a key synthetic enzyme aralkylamine N-acetyltransferase (AANAT) (1). This signaling pathway is shown in Fig. 1. AANAT in turn converts serotonin (5-HT) from a constitutive pool to generate N-acetylserotonin (NAS), from which hydroxyindole-O-methyltransferase generates melatonin (1). The secretion of melatonin and NAS can be increased 100-fold at night (2). In addition to 5-HT, many other neurotransmitters are present elsewhere in the PG, including acetylcholine (ACh), glutamate, GABA, and neuropeptides (3). Their roles in modulating the activity of pinealocytes and melatonin synthesis remain to be established. Here we focus on modulatory effects of 5-HT.Open in a separate windowFig. 1.Synthesis and release of 5-HT and melatonin in rat pinealocytes. The enzymatic pathway from L-tryptophan to 5-HT and melatonin uses four enzymes. AANAT is regulated by adrenergic signaling. Two possibilities are shown (red dotted arrows) for the mechanism of release of 5-HT from pinealocytes: through vesicular exocytosis or through monoamine transporters running in reverse (orange). Released serotonin activates serotonin receptors (blue) on the plasma membrane of pinealocytes. We are suggesting that the receptors up-regulate AANAT acting as an auto- or paracrine mechanism to promote melatonin synthesis. AADC, aromatic amino acid decarboxylase; HIOMT, hydroxyindole-O-methyltransferase; TPH, tryptophan hydroxylase.Serotonergic signaling plays diverse roles in the CNS and peripheral tissues. It is critically involved in mood control, the sleep–wake cycle, breathing, locomotion, and more (4, 5) and acts through 14 distinct, mostly G protein–coupled 5-HT receptors. In the mammalian PG, 5-HT content shows a circadian rhythm, higher during daytime and somewhat lower at night while melatonin is being produced (6, 7). Compared to melatonin release, the 5-HT release from the gland shows only modest (±twofold) circadian variation, continuing in appreciable amounts throughout (8–10). The large pineal pool of 5-HT is continually turning over through constitutive synthesis and secretion (11). Exogenous NE increases 5-HT production and secretion in PGs partly by elevating tryptophan hydroxylase activity (10, 12–17).Secretion of 5-HT from the PG is larger than the elevated nocturnal release of melatonin (9, 10), but the physiological significance of this secretion has not been clear. Released 5-HT is suggested to prime the cAMP-dependent activation of AANAT for melatonin synthesis (17–20). We tested the hypothesis that 5-HT secretion acts as an autocrine signal to promote melatonin synthesis and secretion (Fig. 1) by answering the following questions: What is 1) the secretory mechanism, 2) the type of 5-HT receptor used, and 3) the effect of 5-HT on melatonin secretion? Using optical, electrical, and mass spectrometric (MS) measurements, we found that 5-HT is released from pinealocytes via a nonconventional mechanism and sensitizes NE-induced melatonin synthesis and secretion by activating 5-HT_2C receptors present in the PG. We show that 5-HT qualifies as an autocrine transmitter in the PG.     

Role of extracellular calcium on the regulation of melatonin synthesis in the Syrian hamster pineal gland

The role of extracellular calcium on the induction of cyclic AMP production, N-acetyltransferase (NAT) activity and melatonin synthesis, induced by forskolin, was investigated in Syrian hamster pineal glands. Pineals were removed from hamsters killed either in the first half of the normal dark period or late in the dark period. Forskolin immediately increased NAT activity and melatonin levels only when the glands were collected late in the dark period, while in the first half of the dark phase, hamster pineals responded to forskolin with an increase of NAT activity and melatonin production only after 6 hr of incubation. The absence of calcium prevented the induction of melatonin synthesis by forskolin only when glands were collected early in the dark phase and incubated for 6 hr. In the second half of the normal dark period removal of calcium markedly decreased NAT activity and melatonin levels in glands incubated with forskolin for either 4 or 6 hr. However, the absence of extracellular calcium had no significant effect on the induction of cyclic AMP production by forskolin in pineals collected either early in the dark period or late in the dark phase. These data indicate that at least part of the action of extracellular calcium is indirect and that it affects steps in the induction of melatonin synthesis beyond the accumulation of cAMP.     

Day/night differences in stress-induced gastric lesions in rats with an intact pineal gland or after pinealectomy

Abstract:  The formation of acute gastric lesions depends upon the balance between the aggressive factors promoting mucosal damage and the natural defense mechanisms. Previous studies have shown that melatonin inhibits gastric acid secretion, enhances the release of gastrin, augments gastric blood flow (GBF), increases the cyclooxygenase-2 (COX-2)–prostaglandin (PG) system and scavenges free radicals, resulting in the prevention of stress-induced gastric lesions. Besides the pineal gland, melatonin is also generated in large amounts in the gastrointestinal tract and due to its antioxidant and anti-inflammatory properties; this indole might serve as local protective endogen preventing the development of acute gastric damage. The results of the present study indicate that stress-induced gastric lesions show circadian variations with an increase in the day time and a decline at night. These changes are inversely related to plasma melatonin levels. Following pinealectomy, stress-induced gastric mucosal lesions were more pronounced both during the day and at night, and were accompanied by markedly reduced plasma melatonin levels with a pronounced reduction in mucosal generation of prostaglandin E₂ (PGE₂), GBF and increased free radical formation and by small rise in plasma melatonin during the dark phase. We conclude that stress-induced gastric ulcerations exhibit a circadian variation with an increase in the day and attenuation at night and that these fluctuations of gastric stress ulcerogenesis occur also after pinealectomy, depending upon the interaction of COX–PG and free radicals, probably mediated by the changes in local gastric melatonin.     

Nocturnal plasma melatonin levels in patients suffering from chronic primary insomnia

Abstract: Polysomnographic sleep patterns and melatonin secretion were investigated in 10 patients (age: 41.3 ± 9.5 years) who suffered from chronic primary insomnia and complained predominantly about difficulties in maintaining sleep and in five healthy controls (age 27.2 ± 0.7 years). Nocturnal plasma melatonin concentrations were obtained hourly, measured by direct radioimmunoassay and statistically compared between insomniacs and controls with age as a covariate. Plasma melatonin levels in the patient group tended to begin increasing earlier in the evening and were significantly (P ± 0.01) lower during the middle of the night (peak value 82.5 ± 26.5 pg/ml) than in the healthy controls (peak value 116.8 ± 13.5 pg/ml). Among the patients, the most severely reduced nocturnal plasma melatonin levels were found in those patients with a history of sleep disturbance lasting for longer than five years (N = 6; age 41.8 ± 11.7 years; duration 15.3 ± 5.9 years; peak value 72.1 ± 25.0 pg/ml); whereas those chronic insomniacs affected for fewer than five years had relatively higher nocturnal levels (N = 4; age 40.6 ± 6.5 years; duration 3.8 ± 1.5 years; peak value 98.2 ± 23.9 pg/ml). These results show that the circadian rhythm of melatonin secretion is disturbed in patients with chronic primary insomnia, and that the nocturnal plasma melatonin secretion is increasingly more affected the longer the patients are unable to maintain a regular sleep pattern.     

Physiological levels of melatonin contribute to the antioxidant capacity of human serum

This work evaluates whether physiological concentrations of the pineal secretory product melatonin contribute to the total antioxidant status (TAS) of human serum. Day and nighttime serum samples were collected from healthy volunteers ranging from 2 to 89 years of age and used to measure melatonin and TAS. Results showed that both melatonin and TAS in human serum exhibited 24 hr variations with nocturnal peak values at 01:00 hr. Moreover, exposure of volunteers to light at night resulted in clear decreases of both TAS and melatonin. Furthermore, when melatonin was removed from sera collected at night, the TAS value of the sample was reduced to basal daytime values. In aging studies, it was found that nocturnal serum values of TAS and melatonin exhibited maximal values during the first four decades; thereafter, these values decreased as age advanced. In 60-year-old individuals, day/night differences in serum melatonin and TAS levels were clearly diminished, by more than 80%, with these differences being completely abolished in older individuals. Our results suggest that melatonin contributes to the total antioxidative capability of human serum. This antioxidant contribution of melatonin is reduced as age advances correlating with the age-related reduction of melatonin.