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

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

The influence of different light irradiances on pineal N-acetyltransferase activity and melatonin levels in the cotton rat, Sigmodon hispidus

Wild-captured cotton rats (Sigmodon hispidus) trapped and tested in September and October exhibited a rapid reduction in pineal N-acetyltransferase (NAT) activity and melatonin levels after exposure to a light irradiance of 300 ωW/cm² during the dark period. The half-time for the depression of both NAT and melatonin was on the order of 2 min. The exposure of cotton rats during darkness to much lower irradiances of light, i.e., 5.0, 0.04, 0.03 or 0.01 W/cm², for 32 min also greatly diminished pineal NAT activity and radioimmunoassayable melatonin levels; however, a light irradiance of 0.005 ωW/cm² failed to significantly depress either the acetylating enzyme or the melatonin content of the pineal gland. The results show that the pineal gland of the wild-captured cotton rat, as judged by NAT activity and melatonin levels, is inhibited even by very low irradiances of light.     

Circadian variations in pinealocytes of the Chinese hamster,Cricetulus griseus

Summary Circadian morphological variations of pinealocytes in the superficial pineal of the Chinese hamster (Cricetulus griseus) were studied using quantitative electron-microscopic techniques. The volume of the nucleus and cytoplasm of pinealocytes exhibited similar circadian variations, with the maximum around the middle of the light period and the minimum during the first half of the dark period. Synaptic ribbons in pinealocytes were classified into three groups, type-1, –2 and –3 synaptic ribbons, which appeared as rods, round or irregular bodies and ring-shaped structures, respectively; a synaptic ribbon index was determined for the respective types. The synaptic ribbon index was expressed as the number of synaptic ribbons in the pinealocyte profile representing the cell size. The type-1 synaptic ribbon index, which was smallest during the second half of the light period, was increased during the dark period. The length of straight or slightly curved rods showed a 24-h change similar to that of the type-1 synaptic ribbon index; the length of the rods was maximal during the first half of the dark period and minimal at the end of the light period. There was no apparent circadian variation in the type-2 synaptic ribbon index. The type-3 synaptic ribbon index was higher during the light period than during the dark period; the index attained zero 3h after the onset of darkness and, thereafter, increased gradually.     

Effect of continuous darkness on circadian morphological rhythms in pinealocytes of the Chinese hamster,Cricetulus griseus

Summary Effects of a short-term exposure to continuous darkness on 24-h morphological variations in pinealocytes in the superficial pineal of the Chinese hamster (Cricetulus griseus) were examined. Pinealocytes contained type-1, -2 and -3 synaptic ribbons (SR), which had a central dense structure showing rod-like, various and ring-like profiles, respectively, and the quantity of each type of SR was expressed by SR index. 24-h changes in the type-1 and type-3 SR indices persisted in darkness and thus may be endogenous in nature. As under alternating light and dark (LD) conditions, the type-2 SR indices were almost constant over a 24-h period under continuous darkness, but the indices were larger in animals under darkness than in those under LD conditions. The 24-h variations in the nuclear and cytoplasmic volumes were abolished after exposing animals to darkness for 7 days, suggesting that these rhythms may be regulated exogenously. The amount of condensed chromatin exhibited a circadian change; this rhythm persisted under darkness. The results suggest that 24-h variations in the nuclear and cytoplasmic volumes in pinealocytes of the Chinese hamster are regulated by mechanisms different from those controlling the rhythms in SR and chromatin, and that the changes in the nuclear and cytoplasmic volumes and chromatin are related to the change in synthetic activity of pinealocytes.     

Peptidergic cells in the mammalian pineal gland. Morphological indications for a paracrine regulation of the pinealocyte

Several neuropeptides are present in the mammalian pineal gland. Most of these peptides, eg neuropeptide Y, vasoactive intestinal peptide, and peptide histidine isoleucine, are located in nerve fibres innervating the gland. In some mammalian species, neuropeptides are also found in cells scattered in the pineal parenchyma. In the rat, bipolar cells immunoreactive for somatostatin are present, just as cells containing mRNA encoding somatostatin can be detected in the gland by in situ hybridisation. In the pineal gland of the European hamster, many cells are immunoreactive for enkephalin. Ultrastructural cytochemical analysis of these cells reveals a pinealocyte morphology. Processes from the opioidergic pinealocytes terminate in the parenchyma between the non-immunoreactive pinealocytes. Some of the processes contain small clear and large dense core vesicles and end in club shaped swellings which make synapse-like contacts with other pinealocytes. The ultrastructural morphology suggests that the opioidergic cells exert a paracrine regulation on other pinealocytes.     

Differential response of pineal melatonin levels to light at night in laboratory-raised and wild-captured 13-lined ground squirrels (Spermophilus Tridecemlineatus)

Pineal melatonin levels were compared in laboratory-raised or wild-captured 13-lined ground squirrels (Spermophilus tridecemlineatus) that were either exposed to 10 h of darkness at night or to light which had an irradiance of 400 μW/cm². In laboratory-born squirrels the period of darkness was associated with a gradual rise in pineal melatonin levels with peak values being reached at 0200 h, 6 h after darkness onset. Thereafter, melatonin levels decreased and were back to low daytime levels by 0800 h, 2 h after light onset. The exposure of laboratory-raised animals to an irradiance of 400 μW/cm² during the night totally prevented the nocturnal rise in pineal melatonin levels in these animals. In wild-captured ground squirrels the period of darkness at night was associated with a rapid rise in pineal melatonin such that by 2200 h, 2 h after lights out, peak melatonin values were already attained; additionally, melatonin levels remained high throughout the period of darkness but returned to daytime values by 0800 h. Exposure of wild-captured squirrels to a light irradiance of 400 μW/cm² during the normal dark period was completely incapable of suppressing pineal melatonin levels. The difference in the sensitivity of the pineal gland of laboratory-raised and wild-captured ground squirrels may relate to their previous lighting history.     

Synaptic ribbons of a mammalian pineal gland circadian changes

Summary Synaptic ribbons (SR) of the mammalian pineal gland are functionally enigmatic. In the present study it is shown that in male guinea-pigs kept under a lighting regimen of 12 hrs illumination (7.00–19.00) and 12 hrs darkness (19.00–7.00) the SR of pinealocytes vary about 25-fold in number over a period of 24 hrs. An increase is found between 15.00 and 6.00 and a decrease between 6.00 and 15.00. Analysis of the intracellular localization and the topographical relationships indicate that SR lie near the cell membrane of pinealocytes throughout the 24 hr period and that they are related to adjacent pinealocytes. A working hypothesis put forward implies that SR represent cell organelles involved in intercellular communication and that it is their function to either enhance the secretory activity of the pineal gland or to establish circuits within the gland between adjacent pinealocytes, similar to neuronal circuits.The skilled technical assistance of Mrs. C. Howe is gratefully acknowledged.     

Mitotic activity and its 24-hour rhythm in the rat pineal gland

Previous studies have yielded equivocal results concerning the 24-hour rhythmicity of mitotic activity in the rat pineal. The aim of the present study was to re-investigate this problem by carrying out three separate 24-hour experiments on alternate days. The results obtained confirm previous findings showing that in the pineal gland of adults mitotic activity is low. On average 22.3 mitotic figures of pinealocytes are seen per pineal gland, corresponding to a mitotic index of 0.2-0.6/1,000 pinealocytes. Mitotic activity is distinctly higher at daytime than at night. The timing of the peaks and troughs differs slightly from experiment to experiment. The majority of observations now indicate that in the rat pineal gland mitotic activity is higher at day time than at night.     

Temporally defined induction of c-fos in the rat pineal

In the rat pineal gland c-fos is transiently and markedly induced after the onset of darkness. The induction is abolished following removal of the superior cervical ganglion and reproduced in ganglionectomized animals by treatment with the adrenergic agonist isoproterenol. These findings show for the first time that induction of c-fos forms an integral part of physiological changes in cellular activity. Expression of the c-fos gene may be functionally linked to the nocturnal changes in pineal indole metabolism.     

Influence of continuous light and darkness on the secretory pinealocytes of<Emphasis Type="Italic">Heteropneustes fossilis</Emphasis>

In an earlier study onHeteropneustes fossilis, evidence of secretory activity in the pinealocytes had been demonstrated at the electron microscopic (EM) level and it was found to exist in two phases: a secretory phase (light cells) and a storage phase (dark cells). In the present investigation,H. fossilis was subjected to artificial photoperiods of continuous illumination and continuous darkness for a period of ten days and the effect on the secretory pinealocytes was studied at the EM level. Marked results were observed within the short period of ten days emphasizing the role of environmental photoperiod on the secretory activity of the pinealocytes. During continuous illuminated phase, both light and dark cells were observed: the light cells showed intense secretory activity and dark cells a storage one. During the dark phase both types of cells were present but in different metabolic states and neither of the cells demonstrated synthetic nor storage activity. Light cells were metabolically active but not secretory active and dark cells showed a necrotic condition. Phagocytotic activity of the dark cells was also seen. Intense neural activity was also observed during exposure to both the artificial photoperiods. The results highlight the role of light on the secretory activities of the pinealocytes of the catfish pineal organ.     

Synaptic ribbons during postnatal development of the pineal gland in the golden hamster (Mesocricetus auratus)

Summary Synaptic ribbons, functionally enigmatic structures of mammalian pinealocytes, were studied during the postnatal development of the pineal gland in the golden hamster (Mesocricetus auratus). On day 4 post partum, synaptic ribbons appear in cells that have already started to differentiate into pinealocytes. Between days 4 and 9, an increase in the number of synaptic ribbons occurs, concomitant with the continuing differentiation of the pineal tissue. Between days 9 and 16, when differentiation of this tissue is almost completed, the number of synaptic ribbons decreases and approaches that characteristic of the adult pineal gland. During development, the synaptic ribbons increase in length, and dense core vesicles are frequently found in the vicinity of these structures. It is assumed that a functional relationship exists between dense core vesicles and the synaptic ribbons, which are considered to be engaged in a certain form of secretory activity of the mammalian pineal gland.Supported by the Deutsche Forschungsgemeinschaft     

The presence of opioidergic pinealocytes in the pineal gland of the European hamster (Cricetus cricetus): an immunocytochemical study

By use of antibodies raised against leu-enkephalin and met-enkephalin immunoreactive, opioidergic bi- and multipolar cells were demonstrated in the pineal gland of the European hamster. Ultrastructural analysis of these opioidergic cells revealed them to be pinealocytes. Processes emerged from the cell bodies and terminated in club-shaped swellings containing many small clear and some larger granular vesicles. Some of the terminals made synapse-like contacts with non-immunoreactive pinealocytes. The presence of the opioidergic pinealocytes strongly indicates that the pineal gland of the European hamster, in addition to its pinealopetal nervous regulation, is regulated by intrapineal peptidergic pinealocytes via a synaptic mechanism. A possible paracrine role of the opioidergic cells must also be considered.     

Cytochemical studies on cytoplasmic granular elements in the hamster pineal gland

Summary The pineal gland of adult golden hamsters (Mesocricetus auratus) was studied by various cytochemical methods at the electron microscopic level: (1) the modified chromaffin reaction specific for 5-hydroxytryptamine (5-HT), (2) argentaffin reaction, (3) zinc-iodide-osmium (ZIO) mixture reaction and (4) acid phosphatase reaction. In the pinealocytes, the dense-cored vesicles (80–160 nm in diameter) show both chromaffinity and argentaffinity, while the population of dense bodies (150–400 nm in diameter) is reactive to ammoniacal silver solution and ZIO mixture but not to the modified chromaffin reaction. After incubation for demonstration of acid phosphatase activity, reaction products are localized in some, but not all, of the dense bodies, in some of the small vesicles in the Golgi region and in one or two inner Golgi saccules. In nerve fibers in the pineal gland, small granulated vesicles are also reactive to the modified chromaffin reaction and ZIO mixture. Based upon these cytochemical results the following conclusions have been reached: (1) dense cored vesicles in the pinealocytes and small granulated vesicles in the nerve fibers of the hamster pineal gland contain 5-HT, and (2) the population of dense bodies in the pinealocytes is heterogenous, some are lysosomes and the others are possibly the granules responsible for the secretion of pineal peptides.Supported in part by a grant from the National Science Council, Republic of ChinaDedicated to Professor Doctor Huoyao Wei on the occasion of his 70th birthday     

Effect of photoperiod on pineal gland volume and pinealocyte size in the Chinese hamster, Cricetulus griseus

Male adult (200-day-old) Chinese hamsters (Cricetulus griseus) raised from weaning under either LD 16:8 or LD 8:16 were used. The pineal gland of the Chinese hamster consists of superficial (major) and deep (minor) components and a continuous, or interrupted, narrow parenchymal stalk interposed between them. The volume of the superficial pineal including the parenchymal stalk is greater under LD 16:8 than under LD 8:16. Under both photoperiods, pinealocytes in the superficial pineal have larger nuclei and more abundant cytoplasm than those in the deep pineal. Nuclei in the superficial pineal appear pale and usually have irregular profiles, whereas those in the deep pineal appear dark and have round profiles. In the superficial pineal, pinealocyte nuclei are larger, paler, and more irregular; and, in addition, nuclear density is lower under LD 16:8 than under LD 8:16. Similar, but less prominent, photoperiod-induced changes occur in the volume of the deep pineal, the size of pinealocytes, and pinealocyte nuclear morphology in the deep pineal. The results indicate that the development and differentiation of pinealocytes in both pineal portions may be advanced under long photoperiods and delayed under short photoperiods, although pinealocytes in the deep pineal may remain not fully differentiated even in adults. Since testicular weights and body weights are similar under both photoperiods, the photoperiod may exert marked influences on the development of the pineal gland without affecting reproductive activity and growth rates of animals.     

l-Aspartate but Not the d Form Is Secreted Through Microvesicle-Mediated Exocytosis and Is Sequestered Through Na+-Dependent Transporter in Rat Pinealocytes

Abstract: Rat pinealocytes accumulate glutamate in microvesicles and secrete it through exocytosis so as to transmit signals intercellularly. Glutamate is involved in the negative regulation of norepinephrine-stimulated melatonin production. In this study, we found that aspartate is also released from cultured rat pinealocytes during the exocytosis of glutamate. The release of aspartate was triggered by addition of KCI or A23187 (a Ca²⁺ ionophore) in the presence of Ca²⁺ and was proportional to the amount of l -glutamate released. Furthermore, the release of aspartate was inhibited by both botulinum neurotoxin type E and L- or N-type voltage-gated Ca²⁺ channel blockers. Bay K 8644, an agonist for the L-type Ca²⁺ channel, stimulated the release of aspartate 2.1-fold. Immunohistochemical analyses with antibodies against aspartate and synaptophysin revealed that aspartate is colocalized with synaptophysin in a cultured pinealocyte. HPLC with fluorometric detection indicated that the released aspartate is of the l form, although pinealocytes also contain the d form in their cytoplasm, corresponding to ～30% of the total free aspartate. Radiolabeled l -aspartate was taken up by the microsomal fraction from bovine pineal glands in a Na⁺-dependent manner. The Na⁺-dependent uptake of l -aspartate was strongly inhibited by l -cysteine sulfinate, β-hydroxyaspartate, and l -serine-O-sulfate, inhibitors for the Na⁺-dependent glutamate/aspartate transporter on the plasma membrane. Na⁺-dependent sequestration of l -aspartate was also observed in cultured rat pinealocytes, which was inhibited similarly by these transporter inhibitors. These results strongly suggest that l -aspartate is released through microvesicle-mediated exocytosis from pinealocytes and is taken up again through the Na⁺-dependent transporter at the plasma membrane. The possible role of l -aspartate as an intercellular chemical transmitter in the pineal gland is discussed.     

Twenty-four-hour changes in pinealocytes,capillary endothelial cells and pericapillary and intercellular spaces in the pineal gland of the mouse

Summary Semiquantitative electron-microscopic observations on the pineal gland of dd-mice were carried out to determine whether 24-h rhythms exist in pinealocytes, pericapillary and intercellular spaces and capillary endothelial cells. Nuclear and cytoplasmic areas of pinealocytes and the area of condensed chromatin in pinealocytes showed inversely related circadian rhythms; the former two increased, whereas the latter decreased, during the light period. The extent of pericapillary and wide intercellular spaces exhibited 24-h changes, with an increase and decrease occurring during the light period and the dark period, respectively. The cross-sectional area of endothelial cells decreased and the number of fenestrae increased during the light period; this was reversed during the dark period. The results suggest that the increase in the nuclear and cytoplasmic areas of pinealocytes, the area of pericapillary and wide intercellular spaces and the number of fenestrae, and the decrease in the area of condensed chromatin and endothelial cells during the light period may be related to an increase in synthetic activity of pinealocytes in the mouse.     

Sexual dimorphism among calbindin-D28K immunoreactive cells in the rat pineal body

Calbindin antibodies have been used in neuroanatomical studies to give excellent cytoarchitecural staining and visualization of a Golgi-like cellular morphology. Calbindin-D28K immunoreactivity used in rat pineal gland as a marker detected two classes of pineal cells. One class of small cells representing exclusively glial cells was strongly immunoreactive, and presented a large variety of individual shapes. The majority were a pyramidal shape with one or more processes while others displayed a cytoplasmic lipid droplet. Some small cells occurred around pericapillary spaces. The second class of calbindin-D28K positive cells corresponding to type II pinealocytes were characterized by their large size and less intensive labelling. Type II pinealocytes were round or rectangular; the nucleus was infolded and large with a prominent nucleolus. These large cells were preferentially distributed in the vicinity of vessels and assembled in a cluster of more than ten cells. The lack of S-100 and myeloperoxidase immunoreactivities in large calbindin-D28K cells excluded their possible characterization as glial cells and mononuclear phagocytes, while their size (>15 m) excluded microglial cells. A sex difference was detected between large calbindin-D28K positive cells. The mean calculated number of large positive cells for males was 6361±1504 (n=8) compared to 2162±1235 (n=7) for females. No significative difference was detected between males and females for small calbindin-D28K positive cells.     

Morphologic changes in the pineal gland of rats exposed to continuous darkness

We examined the pineal structure of rats exposed to constant darkness (DD) at light microscopic level. Two groups of rats were exposed to 12:12 light/dark cycle (LD) or DD from their prenatal ontogenesis and then for 3 months after birth. The gland structure of DD rats was observed to have an active appearance. Some of the observed pinealocytes with light nuclei from DD rats were determined to contain double nucleoli. Nuclear area and perimeter of both dark and light types were greater in rats kept in DD than in LD. Rats exposed to DD had more cells with light nuclei and lesser cells with dark ones than rats kept in LD. No significant differences in nuclear characteristics of intermediate type were found between rats kept in LD and those kept in DD. The activity of mammalian pineal can be altered by light conditions to which the animal is exposed.