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Vitamin A deficiency reduces the responsiveness of pineal gland to light in Japanese quail (Coturnix japonica).

Synthesis of melatonin in pineal gland is under the control of light environment. The recent finding of the presence of rhodopsin-like photopigment (pinopsin) and retinal in the avian pinealocytes has led to a hypothesis that vitamin A is involved in photoresponses of the pineal gland. We have thus analyzed the effect of vitamin A deficiency on the regulatory system of melatonin synthesis in the pineal gland of Japanese quail. Depletion of vitamin A from Japanese quails was attained by feeding them with a vitamin A-free diet supplemented with retinoic acid. In the vitamin A-deficient birds, diurnal rhythm in melatonin production persisted such that the phase of the wave was similar to that seen in the control birds. However, the amplitude of the nighttime surge of pineal melatonin was damped by vitamin A deficiency. When the control birds were briefly exposed to light at night, pineal melatonin dropped to the daytime level. In contrast, only slight decrease was observed in the vitamin A-deficient quails. The light responsiveness was restored after feeding the vitamin A-deficient quails with the control diet for 1 week. These results indicate that vitamin A plays essential roles in maintaining sufficient responsiveness of the avian pineal gland to photic input.

Animals↗

Melatonin rhythm onset in the adult siberian hamster: influence of photoperiod but not 60-Hz magnetic field exposure on melatonin content in the pineal gland and in circulation.

To determine the relationship between pineal melatonin production and its appearance in circulation, the rising phase of the pineal and serum melatonin rhythm was studied in the adult Siberian hamster. Melatonin concentrations increased in the pineal gland and in serum at 1.50 and 1.75 h, respectively, relative to lights off in long days (16 h of light/day) and at 2.00 and 2.75 h, respectively, in short days (10 h of light/day). Thus, a photoperiod-dependent melatonin rise in circulation lagged production by the pineal gland by 0.50 h--a delay of 0.75 h in short-day hamsters versus 0.25 h in long-day hamsters. Following initiation of this rise, concentrations that were typical of the nighttime peak were achieved within 2 h of melatonin rhythm onset, regardless of photoperiod. To determine whether clock control of the rising phase of the melatonin rhythm, in the absence of photoperiod cues, may be disrupted by perturbations in the ambient magnetic field, hamsters in constant darkness were acutely exposed to a 1-Gauss, 60-Hz magnetic field for 15 min or were daily exposed to this treatment for 14 or 21 days. Neither the melatonin rise in pineal content or circulation during subjective night was affected by acute or chronic magnetic field exposures; testes regression similarly occurred in sham and daily magnetic field-exposed hamsters in constant darkness. These findings indicate that magnetic field exposures are unlikely to serve as a zeitgeber for the circadian mechanism that controls onset of the melatonin rhythm; rather, photoperiod is a predominant cue that may differentially regulate the rising phase of melatonin production in the pineal gland and concentration in circulation.

Animals↗

Interleukin-1beta expression in the pineal gland of the rat.

Increasing evidence of the neuroimmunomodulatory role of the pineal gland prompted the present study of pineal gland expression of the immunoregulatory cytokine, interleukin (IL)-1beta. IL-1beta was constitutively expressed in the adult gland, with mRNA levels higher in glands collected during the photophase than in those collected during the scotophase of the light:dark cycle. IL-1beta was up-regulated in pineal cultures, after treatment with either norepinephrine (NE) or interferon (IFN)/lipopolysaccharide (LPS). Although both astrocytes and microglia expressed IL-1beta, important differences were found in the cellular expression of this cytokine under in vivo and in vitro conditions. Increased IL-1beta expression by NE ex vivo and the decline in IL-1 expression at night, when NE levels are elevated, can be explained by immunocytochemical data showing that astrocytes are the predominant cell type expressing this cytokine in vivo, whereas IL-1beta-positive cells are predominantly microglia in pineal explants and dispersed cell cultures. These results are consistent with the hypothesis that cytokines secreted by pineal glia (astrocytes and microglia) may have an important regulatory role in the pineal gland.

Animals↗

Stimulatory effect of isoproterenol but not of dibutyryl cyclic AMP on N-acetyltransferase activity and melatonin content of Syrian hamster pineal gland in organ culture.

The purpose of this study was to compare the response of Syrian hamster pineal glands in organ culture either to isoproterenol, a beta-adrenergic agonist, or to dibutyryl cyclic AMP. When pineal glands were collected at night, hamsters were exposed to light for 30 min to depress pineal N-acetyltransferase (NAT) activity and melatonin values to low levels. Pineal glands were removed and placed in organ culture containing either isoproterenol or dibutyryl cyclic AMP and subsequent changes in NAT activity and melatonin levels were measured. At night, isoproterenol (10(-7) or 10(-6) M) induced an increase in the NAT activity and melatonin levels in both pineals and culture media. However, dibutyryl cyclic AMP was either ineffective or minimally effective in stimulating these parameters at either different incubation times (2, 4, and 6 h) or drug concentrations (0.1, 0.5, and 1.0 mM). Conversely, when rat pineal glands were incubated with either isoproterenol (10(-7)) or dibutyryl cyclic AMP (0.5 mM) dramatic rises in NAT activity and melatonin levels were observed. In another experiment, hamster pineal glands were collected from animals killed either late in the light period (19.00 h) or in the latter half of the dark period. Isoproterenol promoted NAT activity and melatonin production only in glands collected in the latter half of the dark phase.

Acetyltransferases↗

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

Production of melatonin in the pineal gland of inbred mice such as C57B1/6J, BALB/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.

Animals↗

[Ca(2+)+Mg2+]-dependent ATPase activity in rat pineal gland.

[Ca(2+)+Mg2+]-dependent ATPase activity in the pineal gland of the rat was examined. The enzyme possesses an apparent Km (Ca2+) of 0.23 microM, and moderately high affinity for Mg2+ and ATP (Km = 53.2 microM and Km = 86.4 microM, respectively). The ATPase activity is sensitive to low concentrations (I50 approximately 1 microM) of vanadate, which specifically inhibits Ca(2+)-ATPase in the plasma membranes of the erythrocyte, cardiomyocytes and synapses. The calmodulin antagonist trifluoperazine reduced significantly Ca(2+)-stimulated, Mg(2+)-dependent ATP hydrolysis. The [Ca(2+)+Mg2+]-dependent ATPase in rat pineal gland exhibits very high affinity for Ca2+, is highly vanadate sensitive and appears to require calmodulin. The enzyme is similar to the Ca(2+)-ATPase of the erythrocyte, cardiomyocytes and synaptic plasma membranes. These new findings may help to elucidate the mechanisms of intracellular calcium homeostasis and the effect of the enzyme on the synthesis of melatonin in the pineal gland.

Adenosine Triphosphate↗

Wavelength dependency of light-induced changes in rhythmic melatonin secretion from chicken pineal gland in vitro.

Avian pineals show rhythmic, circadian melatonin secretion pattern also in vitro. The phase of this rhythm can be modified by changing the illumination. Reversed in vitro illumination reverses the phase of the circadian melatonin rhythm in two days. In the present study the effect of the wavelength on the speed of the phase shift of the melatonin rhythm of the explanted chicken pineals was investigated in a dynamic in vitro bioassay. Chicken pineals were placed into perifusion columns and the tissue was exposed to reversed illumination through various light filters. Changes in the melatonin release during 4 day long experiments were studied. Clear differences in the speed of the reversal of the melatonin rhythm were observed as a function of the transmission spectrum of the light filters. The shorter the transmitted wavelength, the more rapid the phase shift was found. These data are in good agreement with earlier studies showing that the chicken pineal photo-pigment, pinopsin, is a blue light sensitive molecule. Our observation reveals that the blue light sensitive pigment is not only present but also fully functional in controlling the circadian biological clock in the chicken pineal gland.

Journal Article↗

Effects of diazepam administration on melatonin synthesis in the rat pineal gland in vivo.

The effect of diazepam (DZP) on melatonin synthesis in rat pineal gland was investigated in vivo. Subcutaneous injection of DZP (3 mg/kg) 1 h before the start of darkness significantly suppressed nocturnal elevations of pineal N-acetylserotonin (NAS) and melatonin contents in rats, and caused a 2-h delay in reaching the maximum melatonin level in the dark phase. DZP treatment also markedly suppressed the dark-induced increase of pineal N-acetyltransferase activity, which catalyzes the rate-limiting step in melatonin synthesis, but had no effect on hydroxyindole-O-methyltransferase activity, which catalyzes the final step of melatonin formation. Pineal norepinephrine and dopamine contents, in contrast, were not altered by DZP injection. The distribution rate of DZP to the brain reached the highest level 30 min after a single injection, while that to the pineal gland was observed 5 h later (i.e., 4 h after the start of darkness). It is clear that the inhibitory effect of DZP on melatonin synthesis in rat pineal gland appears concomitantly with the increase in the distribution volume of DZP into this gland. These results suggest that the inhibitory effect of DZP on melatonin synthesis results from the drug's direct action on the rat pineal gland.

Acetylserotonin O-Methyltransferase↗

A histochemical study of the adrenergic innervation of the rat pineal gland: evidence for overlap of the innervation from the two superior cervical ganglia and for sprouting following unilateral denervation.

The rat pineal gland receives sympathetic innervation, via the right and left internal carotid nerves, from neurons whose cell bodies are located in the two superior cervical ganglia. Using fluorescence microscopy, we have examined the density and distribution of fluorescent profiles in the pineal gland after lesioning of the internal carotid nerves. Thirty-two hours after sectioning both internal carotid nerves, the density of the fluorescent profiles was 3% of that seen in sham-operated controls, indicating that the lesioned fibers had degenerated. Thirty-two hours after sectioning one internal carotid nerve, the density of the processes was decreased to approximately 50% of the control value. The magnitude of the decrease following a unilateral lesion was similar on both the right and left halves of the gland. Thus, the results suggest that each internal carotid nerve innervates both sides of the pineal gland. The implications of this overlap in the innervation from the two internal carotid nerves for recovery of pineal function after a unilateral lesion are discussed. Sections of pineal glands were also analyzed at later time points after a unilateral lesion. Two weeks after cutting one internal carotid nerve, the density of the fluorescent profiles had increased to greater than 80% of the control value. When the contralateral internal carotid nerve was cut 2 weeks after a unilateral lesion and the pineal gland was examined 32 h later, the density of the fluorescent profiles had decreased to 2% of the sham value. This suggests that all of the compensatory increase in adrenergic processes that takes place following the unilateral lesion is due to sprouting of the contralateral internal carotid nerve rather than to regeneration of the lesioned internal carotid nerve or to sprouting and ingrowth of other adrenergic neurons. It remains to be determined what, if any, functional significance this sprouting has, since the neurally dependent circadian rhythm in serotonin N-acetyltransferase activity in the pineal gland is restored to normal within 32 h after a unilateral lesion; that is, before significant sprouting has occurred.

Acetyltransferases↗

Annual variations of the NPYergic innervation of the pineal gland in the European hamster (Cricetus cricetus): a quantitative immunohistochemical study.

A prominent innervation of the pineal gland of the European hamster with nerve fibres containing neuropeptide Y (NPY) and tyrosine hydroxylase (TH) was demonstrated by means of immunohistochemistry. Nearly all the TH- and NPY-immunoreactive nerve fibres in the superficial pineal gland disappeared after bilateral superior cervical ganglionectomy, showing that the majority of NPY- and TH-immunoreactive nerve fibres belonged to the sympathetic nervous system. Since, in the European hamster, preliminary studies of the NPY-fibre density in the pineal gland had indicated seasonal changes, the density of NPY-immunoreactive nerve fibre profiles was ascertained in the superficial pineal gland in a series of animals between the first part of November and late April. The highest density of NPY-immunoreactive nerve fibre profiles was observed during midwinter. On the other hand, during the same period of the year, the number of sympathetic TH-immunoreactive sympathetic nerve fibre profiles did not exhibit seasonal variation, nor did substitution of testosterone, during the sexually inactive period, affect the density of NPY-containing nerve fibres in the gland. Our results show the presence of a testosterone-independent annual variation in the content of NPY in the sympathetic nerve fibres innervating the pineal gland of the European hamster. This variation can be correlated with the changes in the daily pattern of melatonin production observed by others in the same species at this period of the year.

Animals↗

[The change of the weight of the pineal gland by formalin-fixation (author's transl)].

The weights of 94 human pineal glands were submitted a 3-day-fixation in 10 per cent neutralized formalin, resulting in a loss in weight in one half of the pineal glands and in an increase in weight in the other. After fixation of 4 weeks the difference between actual and initial weight had diminished, only 10.6 per cent of the pineal glands showed a difference larger than +/- 10 per cent. Therefore, the weights of formalin-fixated pineal glands may be used in statistical comparisons. The question why the weights change differently cannot be answered by us.

Adult↗

Comparative view of pineal gland morphology of nocturnal and diurnal birds of tropical origin.

Although having a similar developmental pattern, the pineal gland of tropical birds varies in shape, size, and morphology, probably more than any other part of the avian brain. Following the old classification, we noted a solid follicular (transitional) type of the pineal gland in the nocturnal bird Athene brama, and a tubulo-follicular and elongated tubular types of pineal gland in diurnal birds Perdicula asiatica and Euroloncha punchulata, respectively. Detailed light (LM) and electron microscopic (EM) studies of the pineal gland from these tropical birds revealed the presence of a well-developed, functionally active gland in nocturnal birds (contrary to reports available until now). Unlike diurnal birds, the nocturnal bird A. brama has no deep pineal in the posterior region (near the habenular commissure). It could be that the deep encephalic receptors have no/fewer functions in nocturnal birds. At present, we were unable to define the significance of deep pineal in these tropical avian species. A notable difference in the proximodistal orientation of intrapineal follicles and parenchymatous cells was noted among these birds due to different habitats. Ultrastructurally, the pinealocytes exhibited great similarities in terms of secretory organelles, except for the presence of some peculiar membranous structure in E. punchulata. The pinealocytes have rudimentary photoreceptive features (e.g., outer segment) along with cytoplasmic organelles for secretory activity, suggesting both photosensory and photosecretory types of function. The present study also suggests more heterogenicity in pineal gland morphology (cellular architecture) among diurnal birds than the nocturnal one.

Animals↗

Isoproterenol-stimulated melatonin production by perifused rat pineal glands: age- and time-related effects.

OBJECTIVES: The purpose of this study was to investigate and compare the effects of the beta-adrenergic agonist, isoproterenol, on induced increase in melatonin production in the pineal gland of young and old rats, at different circadian stages. MATERIALS AND METHODS: We report here the effects of 10(-6) M isoproterenol-stimulated melatonin production by perifused pineal glands obtained from young (55 day old) and old (21 month old) male Wistar rats acclimatised to light:dark cycles regimen of 12:12 for 3 weeks. Pineal glands were collected at different circadian stages: 3, 7, 11, 15, 19 and 23 hours after light onset (HALO), and perifused for 510 min. RESULTS: The basal levels of melatonin production in the young rats were approximately twice greater than those of the old rats. Isoproterenol stimulated melatonin production in both young and old rat pineal glands, whatever the circadian stage. The intensity of the response to 10(-6) M isoproterenol infusion was greater in young than in old rat pineal glands (P<0.001), with a trend towards an increase during the light phase, at 7 HALO, in both young and old rat pineal glands, although this trend towards increased melatonin response did not reach statistical significance (P>0.05). CONCLUSIONS: These results show that isoproterenol is able to stimulate both young and old rat pineal glands whatever the circadian stage. The magnitude of isoproterenol stimulation is greater in young than in old glands. Our results also suggest that the pineal gland response to isoproterenol is not dependent on circadian stage, at least, under our experimental conditions.

Adrenergic beta-Agonists↗

Pineal glands of immature rats: rise and fall in N-acetyltransferase activity in vitro.

N-acetyltransferase activity in the pineal glands of immature rats (12--14 days old) shows a spontaneous rise and fall when the glands are placed in organ culture. The peak of the 28-fold change occurs 5--16 hr after the cultures are initiated. This observation can be interpreted in two ways: (1) the pup pineal gland is responding to norepinephrine released when nerve endings degenerate in culture, or (2) the pup pineal gland has an innate ability which is responsible. Whatever the mechanism, the phenomenon is associated with the development of pineal gland function since pineal glands of adult rats do not show a spontaneous rise and fall in identical experiments.

Acetyltransferases↗

[Late effects of experimental ablation of the pineal gland on ultrastructural morphometric changes in thymic epithelial cells].

The late effect of surgical ablation of pineal gland on the morphometric changes in epithelial cells of rat thymus were investigated. The aim of this study is to determine a possible existence of sex-different changes in composition of the epithelial cell component unit long after pinealecotmy what could be important for the subtle understanding of mutual correlation between pincal body and thymus. This article presents results of stereological ultrastructure parameters of thymuscortical and medullar epithelial cells of male and female rats two month after pinealectomy. The experimental animals were divide into two groups: an experimental one (pinealectomized) and the control group (shampinealectomized). Pinealectomized animals were submitted to surgical ablation of pineal gland while the control were undertaken the same surgical treatment but without removal of the pineal gland. Animals were sacrificed 60 th days following the surgery treatment. Parasagital pieces of thymus tissue were fixed by means of immersion in glutaraldehyde and prepared for transmission electron microscopy. Using Weibls multipurpose test system and multilevel sampling technique on electron micrographs the volume and surface density of nucleus (Vu, Sv) and cytoplasm of cortical and medullar thymus epithelial cells were calculated. At the different magnification level were established the volume and surface density of mitochondria (Vvm, Svm) endoplasmic reticulum (Vvr, Svr), vacoule(Vuv) as well as numerical density of mitochondria (Nvm). Our analysis has conformed statistically significant increase in Vv of reticulum and vacuole in both sex of pinealectomized rats. Sv of plasmalema, reticulum and mitochondrial membrane are markedly increased in thymus medullar epithelial cells of pienalectomized rats. Vv of mitochondria is significantly increased in cortical epithelial cells of pienalectomized animals. Results allow us to confirm that mutual correlation between pineal gland and thymus exists but present findings seem to support the concept of sex independent inhibitory action of pineal gland on thymus cortical and medullar epithelial cells.

Animals↗

Bovine and human pineal glands contain substantial quantities of endothelial cell stimulating angiogenic factor.

Using a quantitative assay the amount of a low Mr endothelial cell stimulating angiogenesis factor (ESAF) has been determined in human pineal glands and bovine brain, retina, pineal gland, liver and kidney. Pineal glands contain approximately ten times as much ESAF as the retina or grey and white matter from the cerebral cortex and a hundred times as much as highly vascular tissues such as liver and kidney. The relevance of these findings to the highly vascular nature of the pineal gland is discussed.

Angiogenesis Inducing Agents↗

Demonstration of nerve fibers containing the C-terminal flanking peptide of neuropeptide Y (CPON) in the pig pineal gland (Sus domesticus): an immunocytochemical study by light and electron microscopy.

BACKGROUND: The pineal gland is richly innervated by the neuropeptide Y (NPY)-positive nerves that mostly exibit immunoreactivity for the enzyme tyrosine hydroxylase, a marker of sympathetic nerve fibers. NPY is synthesized as a part of larger prepromolecule. The present study was undertaken to demonstrate that the posttranslational processing of preproNPY resulted in the presence of C-terminal flanking peptide of NPY (CPON) in nerve fibers of the pig pineal gland. METHODS: An immunohistochemical anti-CPON technique was done over mounted sections of perfusion-paraformaldehyde-fixed material. An immunocytochemical preembedding technique was done to study the CPON-positive nerve terminals under electron microscopy. RESULTS: The pig pineal gland is densely innervated by CPON-immunoreactive nerve fibers. These nerve fibers follow from the pineal capsule into the connective tissue septa and farther into the pineal parenchyma, where the varicose branches terminate between the pinealocytes. The fiber density was the highest in the peripheral and ventral parts of the gland. At the ultrastructural level, the CPON-immunoreactive nerve terminals were found in the perivascular spaces and in the parenchyma. The terminals contained small vesicles (30-40 nm in diameter), some of which showed an eccentrically located dense core. In addition, large clear vesicles (80-100 nm in diameter) were present. Some of the CPON-immunoreactive nerve terminals were found in close apposition to the pinealocyte cell membrane, making a synapticlike contact with the pinealocytes. CONCLUSIONS: Our results show the presence of dense CPON-IR innervation in the pig pineal gland. The ultrastructural localization of CPON-IR nerve terminals shows that the peptide can be released to both perivascular and intercellular spaces. The functional role for this peptide in pig pineal gland is still an open question.

Animals↗

Opioidergic innervation of the tree shrew pineal gland: an immunohistochemical study.

The tree shrew (Tupaia glis) has been described as a missing link relating primate to insectivore stock. The pineal gland of the tree shrew consists of a superficial pineal and a deep pineal, which are connected by a long and slender pineal stalk. A monoclonal antibody against leu-enkephalin was used in an immunohistochemical investigation of the tree shrew pineal gland. A moderate innervation of leu-enkephalin immunoreactive nerve fibers has been demonstrated in both superficial and deep pineal gland of the tree shrew. The density of the nerve fibers was slightly higher in the superficial pineal than that of the deep one. The number of immunoreactive nerve fibers were observed in the capsule of the pineal gland from where they entered the pineal parenchyma. Only a few immunoreactive fibers were found in the habenular area and the area rostral to the pineal recess, connecting the habenula and the deep pineal. Furthermore, some positive fibers were located in the pineal stalk. There was no evidence of leu-enkephalin immunoreactive intrapineal cells as seen in the other species of mammal. Therefore, the interspecies variation of opioidergic innervation among the mammals may exist. The lack of intrapineal perikarya is interpreted to indicate that the sources of leu-enkephalin nerve fibers were outside the gland. The anatomical location of the leu-enkephalin immunoreactive nerve fibers in the tree shrew pineal gland supports to both central and peripheral pinealopetal pathways in this species.

Animals↗