Melatonin production by the guinea pig pineal gland in vitro

The fluid bathing pineal glands isolated from guinea pigs was collected serially and its melatonin content was estimated. Production was found to be high (500 pg/5 min) soon after isolation of the gland: it declined exponentially to a maintained lower level (50 pg/5 min) with a half-time of 16-20 min. A rapid increase could be produced by exposure of the gland to the beta-adrenoceptor agonist orciprenaline or by stimulating it electrically. The time course and the extent of the responses to either form of stimulation were similar in glands that had been taken in the morning, at dusk or in mid-dark: the prestimulation peak was lower from glands taken at dusk. Thus the pineal gland of the guinea pig is capable of responding rapidly to stimulation of its beta-adrenoceptors at any time. These responses parallel the depolarization observed intracellularly in this species when the pineal gland is stimulated electrically or exposed to beta-adrenoceptor agonists.     

Melatonin secretion from goldfish pineal gland in organ culture

Pineal glands were removed from goldfish reared under 12L-12D at 25 degrees for 2 weeks. These were incubated for 6 days under (1) normal 12L-12D (lights on 0600-1800 hr), (2) reversed 12L-12D (lights on 1800-0600 hr), (3) continuous dark, or (4) continuous light condition at 25 degrees. The incubation medium was changed at 12-hr intervals (0600-1800 and 1800-0600 hr) and secreted melatonin (MLT) was measured by RIA. Under 12L-12D or reversed 12L-12D, MLT secretion was active in the dark phase and was suppressed in the light phase of a given photoperiod. Under a continuous dark condition, a large amount of MLT was secreted into the medium, although the amount gradually decreased. The MLT secretion was more active in the period corresponding to the dark phase of the acclimatory photoperiod than in the period corresponding to the light phase. This pattern in secretion remained for 4 days. Under a continuous light condition, MLT secretion was suppressed, but the secretion was rapidly increased after changing the photoperiod from the light to the dark condition. These findings clearly indicate that MLT secretion in the organ-cultured pineal gland is photosensitive. It is active under dark and inactive under light conditions. The existence of a circadian rhythm in MLT secretion is also suggested.     

Melatonin synthesis and melatonin-membrane receptor (MT1) expression during rat thymus development: role of the pineal gland

To gain insight into the relationship between thymus and pineal gland during rat development, the melatonin content as well as the activity and expression of the two key enzymes for melatonin biosynthesis, i.e. N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT), were studied in the thymus at fetal and postnatal stages. Moreover, melatonin-membrane receptor (MT1) expression was also analyzed. We found both the expression and activity of thymic NAT and HIOMT at 18 days of fetal life. Additionally, there is production of melatonin in the thymus as well as MT1 expression at this fetal age. These results show values higher in day-time than at night-time. The pineal gland begins to produce significant levels of melatonin around postnatal day 16, and this synthesis shows a circadian rhythm with high values during the dark period; therefore the nocturnal serum melatonin may inhibit thymic melatonin production. To document this, we report an increased melatonin content of the thymus in pinealectomized rats compared with sham-pinealectomized. In conclusion, these results show, for the first time, the presence of the biosynthetic machinery of melatonin and melatonin production in developing rat thymus and that the pineal gland may regulate this process.     

Neurotrophic effects of the pineal gland: Role of non-neuronal cells in co-cultures of the pineal gland and superior cervical ganglia

Abstract: The pineal gland (PG) is a source of several trophic factors. In this study, PG and superior cervical ganglia (SCG) from Sprague-Dawley neonates (1-day-old) were co-cultured to test the hypothesis that endogenous release of PG NGF (or an NGF-like cytokine) is sufficient to promote survival of SCG neurons. Neuronal density of SCG neurons was significantly enhanced when co-cultured with PG for 7 days compared to SCG cultured alone. SCG survival and neurite formation in PG co-cultures was less than in SCG treated with exogenous NGF (100 ng/ml). The neurotrophic effect of PG co-cultures was abolished when 1% anti-NGF was added to the medium. Co-cultures of SCG neurons with established 7-day PG cultures induced extensive SCG neurite formation within 24 hr compared to SCG co-cultured with 1-day PG cultures. This suggests that PG neurotrophic effects are due to PG non-neuronal cells (nnc) that proliferate to confluency by 7 days in culture. S-antigen-positive pinealocytes did not proliferate in culture. There was decreased SCG survival when neurons were seeded onto PG cultures that had been previously killed by drying, which suggests that the neurotrophic effects of nnc are not substrate-dependent. Immunocytochemical characterization of PG nnc revealed a heterogenous mixture of astrocytes, macrophage/ microglia, and fibroblasts. These findings support the hypothesis that NGF is actively secreted by PG and that nnc are the principal source of this neurotophin.     

Melatonin enters the cerebrospinal fluid through the pineal recess.

The pineal recess (PR), a third ventricle (IIIV) evagination penetrating into the pineal gland, could constitute a site of melatonin passage to the cerebrospinal fluid (CSF) and explain the high concentrations of melatonin in this fluid. To test this hypothesis, we characterized melatonin distribution in the IIIV of sheep by CSF collection in the ventral part of IIIV (vIIIV) and in PR. At 30 microl/min collection rate, melatonin concentrations were much higher in PR than in vIIIV (19,934 +/- 6,388 vs. 178 +/- 70 pg/ml, mean +/- SEM, respectively, P < 0.005), and they increased in vIIIV when CSF collection stopped in the PR (P < 0.05). At 6 microl/min, levels increased to 1,682 +/- 585 pg/ml in vIIIV and were not influenced by CSF collection in the PR. This concentration difference between sites and the influence of PR collection on vIIIV levels suggest that melatonin reaches the PR and then diffuses to the IIIV. To confirm the role of PR, we demonstrated that its surgical sealing off decreased IIIV melatonin levels (1,020 +/- 305 pg/ml, compared with 5,984 +/- 1,706 and 6,917 +/- 1,601 pg/ml in shams or animals with a failed sealing off, respectively, P < 0.01) without changes in blood levels. Therefore, this study identified the localization of the main site of penetration of melatonin into the CSF, the pineal recess.     

GABAergic signaling in the rat pineal gland

Pinealocytes secrete melatonin at night in response to norepinephrine released from sympathetic nerve terminals in the pineal gland. The gland also contains many other neurotransmitters whose cellular disposition, activity, and relevance to pineal function are not understood. Here, we clarify sources and demonstrate cellular actions of the neurotransmitter γ‐aminobutyric acid (GABA) using Western blotting and immunohistochemistry of the gland and electrical recording from pinealocytes. GABAergic cells and nerve fibers, defined as containing GABA and the synthetic GAD67, were identified. The cells represent a subset of interstitial cells while the nerve fibers were distinct from the sympathetic innervation. The GABA_A receptor subunit α1 was visualized in close proximity of both GABAergic and sympathetic nerve fibers as well as fine extensions among pinealocytes and blood vessels. The GABA_B1 receptor subunit was localized in the interstitial compartment but not in pinealocytes. Electrophysiology of isolated pinealocytes revealed that GABA and muscimol elicit strong inward chloride currents sensitive to bicuculline and picrotoxin, clear evidence for functional GABA_A receptors on the surface membrane. Applications of elevated potassium solution or the neurotransmitter acetylcholine depolarized the pinealocyte membrane potential enough to open voltage‐gated Ca²⁺ channels leading to intracellular calcium elevations. GABA repolarized the membrane and shut off such calcium rises. In 48–72‐h cultured intact glands, GABA application neither triggered melatonin secretion by itself nor affected norepinephrine‐induced secretion. Thus, strong elements of GABA signaling are present in pineal glands that make large electrical responses in pinealocytes, but physiological roles need to be found.     

Melatonin entrains the circadian rhythm in the rat pineal N-acetyltransferase activity

It has been recently suggested that in mammals the pineal hormone melatonin may be involved not only in transduction of a photoperiodic information, but in a subtle modulation of a phase of the circadian system as well. This suggestion has been based mainly on data about entrainment of the locomotor activity rhythm by melatonin. The present study was undertaken to check whether melatonin may phase-shift also other circadian rhythms, namely the rhythm in pineal N-acetyltransferase activity which is supposed to drive the rhythmic melatonin production in the rat. 50-day-old male Wistar rats were maintained on a regimen of 10 h of light and 14 h of darkness per day. Vehicle or melatonin (1mg/kg rat weight) was injected subcutaneously just before the dark onset. After a single melatonin injection or after administration of melatonin for 5 successive days or after a 4-day treatment with melatonin and a 1-day withdrawal, the evening N-acetyltransferase rise was phase-advanced relative to the rise in rats treated with vehicle only; the phase shift was larger after a repeated than after a single melatonin injection. Under all above-mentioned paradigms of melatonin administration, the morning N-acetyltransferase decline was less phase-advanced than the evening rise. Persistence of the phase advance of the N-acetyltransferase rise after withdrawal of melatonin treatment and a larger phase shift after a repeated than after a single melatonin administration suggest that melatonin may entrain directly a circadian pacemaker controlling the N-acetyltransferase rhythm and affect thus its own rhythmic production.     

The pineal gland and mammalian photoperiodism

The mammalian pineal gland appears to be a major endocrine component in the regulation of photoperiodic responses. The circadian pattern of secretion of the pineal hormone, melatonin, is regulated by the nervous system. Changes in photoperiod, acting via the nervous system, alter the temporal pattern of melatonin secretion. The changes in secretion pattern convey information about daylength from neural components of the circadian system to the reproductive system, and probably to other physiological systems.     

Thymidine kinase activity of the chick pineal gland

Pineal thymidine kinase activity of 1-week-old chicks in situ varied significantly throughout the day. However, the circadian rhythm of thymidine incorporation seen with cultured chick pineal glands was not accompanied by variations in level of thymidine kinase activity in vitro. Thus the circadian rhythm in rate of cumulative incorporation of thymidine by cultured chick pineal glands is not determined by a rhythm in rate of the first reaction of the complex series of reactions by which thymidine is incorporated into DNA.     

Circadian regulation of pineal gland rhythmicity

The pineal gland is a neuroendocrine organ of the brain. Its main task is to synthesize and secrete melatonin, a nocturnal hormone with diverse physiological functions. This review will focus on the central and pineal mechanisms in generation of mammalian pineal rhythmicity including melatonin production. In particular, this review covers the following topics: (1) local control of serotonin and melatonin rhythms; (2) neurotransmitters involved in central control of melatonin; (3) plasticity of the neural circuit controlling melatonin production; (4) role of clock genes in melatonin formation; (5) phase control of pineal rhythmicity; (6) impact of light at night on pineal rhythms; and (7) physiological function of the pineal rhythmicity.     

Prenatal development of the sheep pineal gland: An ultrastructural study

Abstract: The ultrastructure of the pineal gland of 32 sheep embryos was studied from day 54 of development through birth. Embryos were arranged in four age-groups, defined in terms of the most relevant histological features: group 1 (54 to 67 days of prenatal development), group 2 (71 to 92 days), group 3 (98 to 113 days), and group 4 (118 to 150 days). A primary cell type, designated the pinealoblast, was observed from 54 days until birth; ultrastructurally, this cell was found to contain all the organelles required for hormone synthesis. A second cell population, classified as interstitial cells by virtue of their location among pinealoblasts, appeared at 78 days gestation and persisted until birth. Interstitial cells were scarce and exhibited tropism for the perivascular space. From 118 days gestation until birth, a third cell type, termed the pigmented cell, was visible. Pigmented cells, whose ultrastructural characteristics differed from those of pinealoblasts, contained a large number of pigment granules of varying size and shape. The pineal gland of developing sheep embryos showed considerable innervation and abundant vascularization; this, together with certain ultrastructural characteristics, suggests that the gland has a secretory function in uterine life.     

Tissue kallikrein in the rat pineal gland: An immunocytochemical study

Abstract: Tissue kallikrein in the rat pineal gland was immunocytochemically investigated with the aid of specific antiserum against rat urinary kallikrein. We also compared the tissue kallikrein immunoreactivity of the pineal gland with that of the submandibular gland and kidney, which have been well established as tissue kallikrein-synthesizing organs. The cytoplasm of pinealocytes from both the superficial and the deep portion of the gland exhibited specific immunolabeling for tissue kallikrein, but the immunoreaction was weaker than that observed in exocrine organs. Two types of tissue kallikrein-immunoreactive pinealocytes were distinguished; the first predominant type displayed moderate immunostaining, whereas a small number of cells, the second type, were so intensely labeled that their cytoplasmic processes were clearly outlined. The results of the present study suggest the existence of different types of pinealocytes and a potential physiological role of tissue kallikrein in the rat pineal gland.