"Synaptic" Ribbons and Spherules Lacking in the Pineal Gland of the European Hamster Appear After Ganglionectomy

"Synaptic" ribbons and spherules are typical organelles of the mammalian pinealocytes, that present quantitative day-night variations parallel to pineal melatonin levels. The European hamster is a wild photoperiodic mammal whose pineal melatonin concentrations do not vary during the 24 hr light-dark cycle. The aim of this study was thus to see if a rhythmic variation in synaptic structures was nevertheless present in this species. It was found that, in contrast to all of the photoperiodic species studied to date, the European hamster pinealocyte contains no ribbons, and virtually no spherules. Moreover, constant administration of melatonin does not result in the occurrence of these organelles; nevertheless, the pinealocytes have the potentiality to produce spherules and, to a lesser extent, ribbons, as they are observed in ganglionectomized animals and in the deep pineal of some hamsters after ablation of the superficial part of the gland. The formation of pineal synaptic structures, normally lacking in the European hamster, thus seems directly related to the noradrenergic innervation of the gland.     

Effect of superior cervical ganglionectomy on the ultrastructure of the pinealocyte in the cotton rat (Sigmodon hispidus)

The ultrastructure of pinealocytes was examined morphometrically in superior cervical ganglionectomized and sham-operated cotton rats. Following denervation, the pinealocytes decreased in size. Reduced areas of the nucleoli, Golgi apparatus, granular endoplasmic reticulum, mitochondria, and vacuoles containing a flocculent material as well as a decreased number of dense-core vesicles were noted. In contrast, the numbers of “synaptic” ribbons and ribbon fields increased. The nature of ultrastructural changes observed in the cotton rat pinealocyte after sympathectomy may indicate a diminution of the presumptive secretory processes of this cell. The presence of a small number of myelinated and unmyelinated axons in the pineal gland of the cotton rat following superior cervical ganglionectomy suggests that the sympathetic nerve fibers from the superior cervical ganglia do not comprise the only source of innervation to the pineal. Additionally, the presence of striated muscle fibers has been observed in the present study in the pineal gland of the cotton rat which heretofore has not been reported in this species.     

Effects of Isoproterenol on Synaptic Ribbons in Pinealocytes of the Rat and C57BL/6J Mouse

Synaptic ribbons (SR) in melatonin-deficient pinealocytes of the C57BL/6J mouse were quantitatively compared to SR in pinealocytes of the rat after beta-adrenergic receptor activation by isoproterenol. Two populations of SR comprising synaptic spherules (SRsp) and synaptic rods (SRr) were described in both the mouse and the rat, but species differences existed in the ratio of SRr to SRsp. Isoproterenol caused a significant increase in frequency of SR of the rat but had little or no effect on SR populations in the mouse. It is unlikely that beta-adrenergic receptors are absent on mouse pinealocytes or were not activated since isoproterenol elevated plasma renin concentrations indicating activation of beta-adrenergic receptors. Furthermore the pineal of both species receives heavy sympathetic input. These findings indicate that the role and regulation of pinealocyte SR are complex and are functionally linked to beta-adrenergic receptors as well as other mechanisms related to the production of melatonin.     

Structure of the pineal gland in the adult cat

Abstract: The ultrastructure of the pineal gland in the adult cat is described and compared with that of other mammals. Connective tissue spaces showed capillar-ies with nonfenestrated endothelia and numerous unmyelinated nerve fibers. In the proximal region of the gland, myelinated nerve fibers coming from the anterior commissure were also found. Cat pinealocytes showed a nucleus with prominent nucleoli, a well developed Golgi apparatus, centrioles, granular endoplasmic reticulum, ribosomes, abundant microtubuli and enlarged mitochondria. Pinealo-cytes showed several long processes with bulbous endings filled with clear vesicles and scarce "synaptic" ribbons. Pineal astrocytes and their processes were characterized by the presence of abundant filaments.     

No Correlation of Pineal "Synaptic" Ribbon Numbers and Melatonin Formation in Individual Rat Pineal Glands

As previous circadian studies of pineal "synaptic" ribbon numbers and melatonin formation suggested that a positive correlation of the two variables exists, in the present investigation this problem was examined in individual pineal glands of rats killed at 1200 h and 2400 h, respectively. For this purpose, one half of the gland was processed for electron microscopy and the ribbons were counted in an area of 20,000 micron2 tissue; in the other half serotonin N-acetyltransferase (NAT) activity and melatonin content were determined. No correlation was found to exist between ribbon numbers and pineal NAT activity, pineal melatonin levels and serum melatonin levels, either at day- or at nighttime. It is concluded that the ribbons may perhaps be more closely related to the innervation of the pineal gland than to melatonin formation.     

Lack of "Synaptic" Ribbons in the Pineal Gland of BALB/c Mice

In mammalian pinealocytes "synaptic" ribbons (SR) are regularly occurring organelles that are functionally poorly understood. Since in a number of studies on the mouse pineal gland the presence of SR has not been mentioned, it was the aim of this investigation to quantitate SR in mice. BALB/c mice were chosen, which have recently been shown to have a genetic defect for melatonin synthesis. The pineals of 15 mice killed at night, when SR numbers are normally high, were examined electron microscopically, scanning an area of greater than 20,000 micron 2 per gland. In none of these pineals were SR detected. It is concluded that the lack or extreme rarity of SR in laboratory mice may be related to the inability of the gland to synthesize melatonin, not to the absence of melatonin per se, as melatonin administration in mice does not result in the occurrence of SR.     

Effect of magnetic field on pineal gland volume and pinealocyte size in the rat

Abstract: Light microscopic observations on the superficial pineal gland of Wistar-King rats were made to examine whether or not pineal volume and pinealocyte size, expressed as nuclear density, at daytime or nighttime are affected by long-term exposure to 50 Hz rotating magnetic field (MF) at 5.0 μT. Determinations of pineal volume and pinealocyte size were repeated twice (April and October) during the year. Size of pinealocytes in MF-exposed and sham-exposed rats exhibited, in addition to the difference between peripheral and central regions, regional differences in a proximodistal direction; pinealocytes in the distal and middle-peripheral regions were usually larger than those in the proximal and middle-central regions at daytime or nighttime. In October, distal and proximal pinealocytes showed significant day-night changes in size in sham-exposed rats, but not in MF-exposed animals. The situations in the two groups were almost reversed in April. Significant day-night differences were scarcely found in pinealocyte size in the middle region in the two groups. Throughout the study, pineal volume and pinealocyte size in each region were generally the same between MF-exposed and sham-exposed rats at daytime or nighttime. The results suggest that pinealocytes in the distal and proximal regions, but not those in the middle region, are affected by MF-exposure; day-night differences in sizes of distal and proximal pinealocytes appear in April and disappear in October under the influence of MF. MF may exert an effect on mechanisms controlling day-night rhythms of pinealocyte size in the rat.     

The Pineal Gland in Newborn Southern Elephant Seals, Mirounga leonina

In the newborn southern elephant seal the pineal gland is very large, and both pineal and plasma melatonin concentration is elevated. The pineal gland was investigated during the first 24 h, and up to 20 days of age, in elephant seal pups. A primary aim of this investigation was to determine whether there are obvious ultrastructural characteristics of pinealocytes that are exhibiting extraordinarily high levels of activity. Blood and pineal glands were collected from thirty seven pups of known age which were sampled at random from early September to early November (1985) at Macquarie Island. The pineal gland is large (mean weight, 4.71 +/- 0.35 gm, range 1-9.3 gm) and actively secreting melatonin at birth. Melatonin concentrations were extremely variable, yet very high in pups during the first 24 h post-partum. Mean melatonin plasma concentration for pups 0-24 h was 17632.8 +/- 5723.8 pmol/l (4090.8 +/- 1327.9 pg/ml), ranging from 126 pmol/l (29 pg/ml) to 297000 pmol/l (68904 pg/ml). Electron microscopic examination did not reveal any marked changes in pinealocyte ultrastructure suggestive of increased secretory activity during this period. The large and extremely active pineal gland in newborn southern elephant seal suggests that it is actively involved in thermoregulation.     

Cytodifferentiation of the Chick Pineal Gland, With Special Reference to the Photosensory and Secretory Elements

Cytodifferentiation of the chick pineal gland throughout the embryonic development was investigated with light and electron microscopy. The chick pineal anlage appears first as a small evagination in the diencephalic roof at 60 h of incubation (27-30 somites). Until day 5 of incubation, pineal anlage cells are undifferentiated and appear similar to ventricular ependymal cells. Subsequently, pinealocytes and supporting cells are first distinguishable at 7-8 days, and parafollicular cells are distinguishable at 12 days of incubation. Pigment-containing cells after 6 days and nerve cells after 17 days of incubation gradually increase, especially in the posterior wall of the pineal recess. During embryonic development, the chick pineal gland has both photosensory and secretory elements: viz. the former, mitochondria-laden apical protrusions, synaptic ribbons, lamellar whorl-like cilia of the pinealocytes, and adjacent appearance of the pigment-containing cells and the nerve cells; and the latter, dense-cored vesicles of the pinealocytes and dense bodies of the supporting cells. Moreover, nuclear invaginations having a large lipid droplet nearby and some aggregations of glycogen are found in the pinealocytes and are transitory changes in structure restricted to certain days of incubation.     

Comparison of the Rat Pinealocyte Ultrastructure With Melatonin Concentrations During Daytime and at Night

The aim of this study was to investigate the ultrastructure of rat pinealocyte during daytime (1600 h) and at night (0100 h) and to compare these observations with serum melatonin levels in the same animals. In addition, pineal melatonin concentrations were determined in other animals. Both serum and pineal melatonin concentrations were significantly higher at night than during daytime (34 and 21 times, respectively). Sizes of pinealocytes, their nuclei, and nucleoli, as well as cross-sectional areas of mitochondria and granular endoplasmic reticulum were also higher at night than during daytime, whereas areas of lysosomes, Golgi apparatus, and vacuoles containing flocculent material did not differ at the time points studied. In contrast, the number of dense-core vesicles was higher during daytime. The results of the present study show that morphological patterns of higher metabolic activity of the rat pinealocyte at night when compared to those during daytime correlate with melatonin concentrations.     

Synaptic Junctions Between the Adrenergic Axon Varicosity and the Pinealocyte in the Rat

Intercellular relationships of sympathetic nerve fibers and cells in pineal glands of Long-Evans rats and yellow-bellied country rats (Rattus losea Swinhoe) were studied with conventional electron microscopy. Typical synapses were found between adrenergic axon varicosities and pinealocytes. The synaptic cleft was 20-30 nm thick and contained granular or filamentous material of moderate electron density. Some small granulated vesicles attached to, or fused with, the presynaptic axolemma, a feature suggesting an exocytotic release of the vesicle content. The occurrence of membranous cisternae or tubules in the synaptic nerve ending may connote the phenomenon of synaptic vesicle recycling. Beneath the postsynaptic pinealocyte plasmalemma there was a dense plaque corresponding to the postsynaptic density of neurons. Possible origins and functional significance of the synapse-making nerve fibers are also discussed.     

Quantitative differences in the pineal ultrastructure of perinatal and adult harp (Phoca groenlandica) and hooded seals (Cystophora cristata)

Seals are unique among mammals in that newborns have a large pineal gland and extremely high plasma levels of melatonin at birth. Melatonin levels are also high in the seal fetus but decline rapidly during the first few days of life. The aim of the present study was to provide quantitative information about the ultrastructure of the seal pineal gland using fetal, newborn, and adult hooded seals (Cystophora cristata), and newborn and adult harp seals (Phoca groenlandica). The relative and absolute volumes of pinealocytes (Pi), arteries and veins, nerves, connective tissue, capillaries and glial cells, as well as mitocondria and lipid droplets in Pi, were calculated by use of point count analysis. Whereas the pineal ultrastructure was similar in fetuses and newborns, both seal species showed a pronounced and particular reduction in the volume of Pi and a similar reduction in pinealocyte mitochondria. There was also a shift from unmyelinated to myelinated pineal nerves in adults compared with fetal/newborns. The selective and marked reduction of Pi may explain the zonated pineal structure typical of the adult seal. The results demonstrate that the fetal gland is as large and active as that of the newborn seal and support the notion that the large size and high activity of the pineal gland in the newborn seal is a fading consequence of its prenatal condition.     

Development of day-night rhythmicity in "synaptic" ribbon numbers in the pinealocytes of posthatch chicks kept under either natural photoperiodic conditions or continuous illumination

Pineal synaptic ribbons (SR) undergo characteristic changes over a period of 24 hr under natural photoperiodic conditions in various vertebrates, being low in number during daytime and elevated at night. During posthatch development of chicks, the rhythmicity of SR numbers is reported to appear at the age of about 2 weeks. Because the influence of external light during the growth phase of chicks on the development of day-night rhythmicity in SR numbers is unknown, we studied day-night differences in SR numbers in the pinealocytes of chicks at the posthatch ages of 15, 17, and 19 days; chicks had previously been kept under natural photoperiodic conditions or continuous illumination. Under natural photoperiodic conditions a statistically significant nocturnal (midnight) rise in SR numbers over the value of midday was seen in the pineal of 17- and 19-day-old chicks, but not in 15-day-old chicks. SR numbers in the pinealocytes of continuously illuminated chicks did not show any day-night rhythmicity on days either 15 or 17, but exhibited significant day-night differences on day 19 posthatch. These findings suggest that continuous illumination, which is known to dampen circadian rhythmicity of melatonin secretion in the chick pineal, causes a delay, but not a total suppression of the mechanism involved in the ontogenic development of diurnal rhythmicity in SR numbers in the pinealocytes of chicks.     

Close microtopographical relationships between sympathetic nerve terminals and bulbous process endings of pinealocytes in the pineal gland of the Mongolian gerbil

Abstract: Previous studies have shown that pinealocytes of the gerbil pineal gland exhibit processes that form terminal swellings filled with abundant electron-lucent microvesicles. The membrane of these presumptive secretory microvesicles is known to contain synaptophysin, a major integral glycoprotein of neuronal synaptic vesicles. The present study was conducted to evaluate the microtopographical relationships between the vesicle-rich process swellings and intra-pineal nerve terminals. For this purpose, both nerve terminals and pinealocyte process endings were visualized immunohistochemically in the same semi-thin sections of plastic-embedded gerbil pineals, using antibodies directed against synaptophysin. This approach consistently revealed close spatial associations of punctate immunopositive nerve endings with intensely stained bulbous process terminals of pinealocytes in or near the perivascular spaces. The light-microscopic observations of intimate neuronal-pinealocytic relationships were corroborated at the electron-microscopic level. Perivascular varicosities with ultrastructural features characteristic of sympathetic nerve terminals were frequently juxtaposed to vesicle-filled process endings of pinealocytes. Analysis of serial thin sections showed that multiple point-to-point contacts are encountered between noradrenergic nerve terminals and pinealocytic process swellings. Our morphological findings imply that bulbous process terminals, at least in the gerbil pineal gland, are major targets for the neuronal control of the secretory activity of pinealocytes.     

Fine structural alterations in the aging rat pineal gland

The pineal glands of 1 mo to 28 mo old male and female rats were examined by electron microscopy. With increasing age the following observations were made: (1) the capsule thickness increases; (2) there is an increase in collagen infiltration; (3) increased amounts of granular deposits are seen between cells; (4) there is a greater variability in the number of light pinealocytes; (5) pinealocytes with nuclear invaginations and pinealocytes with nuclear inclusions increase in number; (6) more cytoplasmic dense bodies are seen in pinealocytes and gliocytes; (7) the maximum diameter of pinealocyte lipid droplets tends to increase; (8) no definite change is observed in granular endoplasmic reticulum; (9) occasional cells are found to contain reticulated mitochondria, and a few cell processes have an appearance similar to neuroaxonal dystrophy. Besides illustrating age-related changes, the present study proves the existence of pinealocyte nuclear inclusions, suggests the possibility of pineal concretions in the rat, and morphometrically or semi-morphometrically analyzes several structural features not previously quantified.     

Fine structural alterations in the aging rat pineal gland

The pineal glands of 1 mo to 28 mo old male and female rats were examined by electron microscopy. With increasing age the following observations were made: (1) the capsule thickness increases; (2) there is an increase in collagen infiltration; (3) increased amounts of granular deposits are seen between cells; (4) there is a greater variability in the number of light pinealocytes; (5) pinealocytes with nuclear invaginations and pinealocytes with nuclear inclusions increase in number; (6) more cytoplasmic dense bodies are seen in pinealocytes and gliocytes; (7) the maximum diameter of pinealocyte lipid droplets tends to increase; (8) no definite change is observed in granular endoplasmic reticulum; (9) occasional cells are found to contain reticulated mitochondria, and a few cell processes have an appearance similar to neuroaxonal dystrophy. Besides illustrating age-rated changes, the present study proves the existence of pinealocyte nuclear inclusions, suggests the possibility of pineal concretions in the rat, and morphometrically or semi-morphometrically analyzes several structural features not previously quantified.     

Pinealocyte subsurface cisterns III: Storage of calcium ions and their probable role in cell stimulation

Different techniques for the ultrastructural demonstration of calcium have been applied to the pineal gland of Meriones unguiculatus, attention being focussed on the endoplasmic reticulum (ER) and its subsurface cisterns (ssc). By means of a "loading" method [Walz, 1982; Wakasugi et al., 1982] it is shown that the pinealocyte ER-ssc system sequesters calcium with dependency on ATP. Furthermore, a modification of the method of Duce and Keen [1978] is presented which turned out a) to be sensitive enough to demonstrate the cell's own low amounts of calcium as fine granular precipitates, and b) to preserve ultrastructure sufficiently. This method rendered possible comparison of the calcium distribution inside pinealocytes of the following groups: animals fixed during daytime, animals fixed at night, animals fixed at night with prior exposure to bright white light, animals fixed at night but injected at the end of the preceding light period with a pharmacon known to prevent the release of calcium from the ER of muscle fibers (Dantrolen). In contrast to the daytime findings, the pinealocyte ER-ssc system at night is free of precipitable calcium; nocturnal illumination induces reacquisition, Dantrolen hinders nocturnal depletion. From the nocturnal coincidence of pinealocyte activity and calcium release from ssc, and from other cytological and experimental data, it is concluded that the functional significance of ssc refers to the regulation of pinealocyte sensitivity. Vice versa, pinealocyte activity may influence ER expansion and ssc size via the calcium-dependent stability of microtubules.     

Pinealocyte Dense-Cored Vesicles and Synaptic Ribbons: A Correlative Ultrastructural-Biochemical Investigation in Rats and Mice

Dense-cored vesicles (DCV) and synaptic ribbons (SR) were quantified in the pineal gland of the rat (Sprague-Dawley) and mouse (Sasco/ICR strain), and day/night differences in frequency of these organelles correlated with levels of indoles determined by high performance liquid chromatography (HPLC). There were significant day/night differences in levels of serotonin (5HT), 5-hydroxyindole acetic acid (5HIAA), N-acetyl-5HT, and melatonin in the rat gland. Melatonin and N-acetyl-5HT were not detectable in the mouse gland sampled every 4 h over the light:dark cycle. The concentrations of 5HT and 5HIAA (ng/microgram protein) were similar in light-adapted rats and mice, but these indoles did not exhibit a circadian rhythm in the mouse gland. Correlative ultrastructural/biochemical results suggest that DCV do not contain physiologically important stores of 5HT since 1) the mouse gland contains the same number of DCV as the rat during the daytime, but only one-tenth the levels of 5HT, 2) day/night 5HT levels do not vary in the mouse gland, but there is a significant nocturnal decline in DCV numbers, and 3) 5HT levels in the rat gland decline at night when DCV numbers increase. Numbers of SR were significantly elevated at night in the rat and mouse, and the frequency of this organelle was similar in both species. However, ribbon-type SR predominated in rat pinealocytes, whereas SR in the mouse were almost exclusively spherical in shape. Day/night differences in SR numbers in the mouse gland suggest that cellular mechanisms regulating the frequency of this organelle do not involve factors related to indole metabolism. Because of the lack of photoperiodic effects on indole metabolism in the mouse pineal gland, this species is a potentially important model to study the functional relationship of pinealocyte organelles to cyclical changes in pineal products other than indoles (e.g., peptide/protein factors).