Ultrastructure of the pineal gland of the eastern chipmunk (Tamias striatus)

The ultrastructure of the pineal gland of the wild-captured eastern chipmunk (Tamias striatus) was examined. A homogenous population of pinealocytes was the characteristic cellular element of the chipmunk pineal gland. Often, pinealocytes showed a folliclelike arrangement. Mitochondria, Golgi apparatus, granular endoplasmic reticulum, lysosomes, centrioles, dense-core vesicles, clear vesicles, glycogen particles, and microtubules were consistent components of the pinealocyte cytoplasm. The extraordinary ultrastructural feature of the chipmunk pinealocyte was the presence of extremely large numbers of “synaptic” ribbons. The number of “synaptic” ribbons in this species exceeded by a factor of five to 30 times that found in any species previously reported. In addition to pinealocytes, the pineal parenchyma contained glial cells (oligodendrocytes and fibrous astrocytes). Capillaries of the pineal gland of the chipmunk consisted of a fenestrated endothelium. Adrenergic nerve terminals were relatively sparse.     

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.     

"Light" and "dark" pinealocytes of the porcine pineal gland

Both qualitative and quantitative comparative studies of "dark" and "light" pinealocytes of the porcine pineal gland have been carried out. These cells differ from each other in their electronic density of cytoplasm, shape of nucleus, the structure of membrane bound dense bodies and the number of microtubules and smooth endoplasmic reticulum. The membrane bound dense bodies--characteristic structures of pig pinealocytes as well dense core vesicles occur in both types of cells. The relative volume of the majority of the cells' organellae apart from the Golgi apparatus, also do not show any significant difference. The results obtained support a functional basis for pinealocyte differentiation in the porcine pineal gland.     

Morphologic development of neonatal rat pinealocytes in monolayer culture

Summary The morphological development of pinealocytes maintained in monolayer culture, without the neural and humoral effects present in the developing rat has been studied and compared with the development that occurs in vivo. Pinealocytes in 5 day cultures contained organelles that were similar to those present in the pineals of intact 5 day old rats. However, light and dark cells were not noted in culture, and the cultured cells did not have the dense granules noted in vivo. As pinealocytes developed in culture, cytoplasmic processes increased in length and number. By 21 days of culture age, synaptic ribbons were found to have decreased in number, the difference between light cell and dark cell cytoplasm had become more prominent, and dense-cored vesicles had become more numerous, just as in the developing gland in vivo. These results suggest that the complex neural and humoral factors impinging upon the developing neonatal pineal in the intact animal may not be necessary for some aspects of its ultrastructural differentiation.     

Electron microscopy of the rabbit pineal organ in vitro

The results presented in this study show that the rabbit pineal organ cultured in vitro retained its in vivo fine structure for at least eight days. However, the Golgi complex in the light pinealocytes stopped forming dense core vesicles while vesicle-crowned ribbons increased in number. After addition of norepinephrine to the culture medium, the Golgi complex once more began the production of dense core vesicles. Terminals of light pinealocytic processes then often contained Golgi dense core vesicles in close contact with the cell membrane, suggesting the release of the vesicular content into the intercellular and perivascular spaces. A close topographical relationship between Golgi dense core vesicles and vesicle-crowned ribbons was observed.     

Prenatal development of "synaptic" ribbons in the guinea pig pineal gland

Pineal "synaptic" ribbons are a heterogeneous population of organelles. "Synaptic" ribbons (SR) sensu stricto, "synaptic" spherules (SS), and intermediate forms (IMF) are present. Their function and origin are unknown, and a knowledge of their prenatal development is lacking. Thus the pineal glands of prenatal, neonatal, and adult guinea pigs were prepared for electron microscopy. "Synaptic" ribbons were studied morphologically and quantitatively. The three categories of "synaptic" ribbons reported in adult pineal glands were also present in prenatal pineal glands. Their structural features, distribution, grouping, and composition patterns are similar to those in adults. "Synaptic" ribbons were first detected in pinealocytes of the distal region of a 42-day postcoitus (PC) pineal gland and were comparable with those in adults. They increased in number with age and reached a peak at 63 days PC, followed by a steep decline at 66 and 67 days PC. By day 69 PC, the numbers increased again and showed a dramatic increase after birth. Several true ribbon synapses were seen at day 63 PC between pinealocyte cell processes or between pinealocyte cell process and pinealocyte cell body. Since true ribbon synapses have not been found in adult guinea pig pinealocytes, their synaptic nature could have been lost during development. No precursors for the "synaptic" ribbons were found. The endoplasmic reticulum cisternae may be the origin for the ribbon vesicles because of their close association with the "synaptic" ribbons.     

The pineal gland of the gerbil,Meriones unguiculatus

Summary Electron microscopy was employed in a study of the pineal gland of the Mongolian gerbil (Meriones unguiculatus). It was determined that the gerbil pineal gland contains pinealocytes and glial cells with the pinealocytes being the predominant cell type. The pinealocytes contain numerous organelles traditionally considered as being either synthetic or secretory in function such as an extensive Golgi region, smooth (SER) and rough (RER) endoplasmic reticulum, secretory vesicles and microtubules. Other cytoplasmic components are also present in the pinealocytes (synaptic ribbons, subsurface cisternae) for which no function has been assigned. Dense-cored vesicles are rare. Vacuolated pinealocytes are present and appear to be intimately associated with the formation of the pineal concertions. Evidence presented supports the proposal that the concretions form within the vacuoles. Once the concretions reach an enlarged state, the vacuolated pinealocytes break down and the concretions are thus extruded into the extracellular space where they apparently continue to increase in size. The morphology of the glial cells was interpreted as indicative of a high synthetic activity. The glial cells contain predominantly the rough variety of endoplasmic reticulum and form an expansion around the wide perivascular area.Supported by NSF grant PCM 77-05734     

Postnatal development of "synaptic" ribbons and spherules in the guinea pig pineal gland

Previous studies have shown that the functionally enigmatic pineal "synaptic" ribbons are structurally a heterogeneous group of organelles consisting of rodlike ribbons sensu stricto, spherules, and intermediate forms. As ribbons and spherules react differently under various experimental conditions, these organelles were studied qualitatively and quantitatively during the postnatal period in guinea pigs. It was found that the pinealocytes were highly differentiated at birth and contained all three forms of "synaptic" structures. Ribbons and intermediate forms were more abundant than spherules and exhibited a striking increase in number on postnatal days 1 and 2; this increase was followed by a distinct trough and by a second peak at days 12 and 13, after which their numbers declined to reach adult levels by day 20. The spherules were small in number at birth and did not show the large immediate postnatal increase observed for the ribbons and intermediate forms. Instead there was a steady numerical increase up to day 12 (absolute number) or day 15 (relative numbers), followed by a decrease to adult level by day 20. Whereas during the early postnatal period (days 1 to 3) the majority of pinealocytes were characterized by ribbons and intermediate forms, with increasing age spherule-bearing pinealocytes increased in number. As ribbons and spherules were usually not found in the same pinealocyte, the present findings are interpreted to mean that ribbons and spherules characterize different types of pinealocytes showing an inverse numerical development postnatally. Developmentally intermediate forms behave like ribbons.     

Circadian changes in microtubules,synaptic ribbons and synaptic ribbon fields in the pinealocytes of the baboon (Papio ursinus)

Summary In baboons kept under controlled lighting conditions, microtubules (MT) are readily seen in the perikaryal cytoplasm and in the perivascular processes of pinealocytes. A significant increase in the number of MT, single synaptic ribbons (SR) and the formation of synaptic ribbon fields (RF, i.e. organelles which consist of multiple dense rodlets or plates, and vesicles), occur during the dark phase of a circadian light-dark cycle. MT may act as tracks for the oriented flow of vesicles derived from the smooth endoplasmic reticulum, to cytoplasmic sites where RF are being formed. The origin of the dense rodlets of RF remains unknown. Structural differences between SR and RF indicate that the latter organelles are not directly involved in impulse propagation between adjacent baboon pinealocytes. RF may function as storage organelles for some of the pineal secretory products which are formed in large amounts during the dark phase.     

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     

Light and drug induced changes of epiphysial synaptic ribbons

Summary In the present investigation experiments were carried out to determine whether the functionally obscure synaptic ribbons of mammalian pinealocytes can be affected by acute changes in environmental lighting and which chemical processes may be involved in their regulation. Experiments carried out in male guinea-pigs have shown that the amounts of synaptic ribbons are immediately affected by changes in the lighting pattern. Extension of the light period reduced the normally occurring increase, whereas extension of the dark period inhibited the normally occurring decrease in the amount of synaptic ribbons. Results following injections of a number of drugs known to influence pineal function (noradrenaline, L-DOPA, propranolol, reserpine and p-chlorophenylalanine, respectively) suggest that synaptic ribbons may be directly or indirectly regulated by -adrenergic mechanisms.Dedicated to Professor Wolfgang Bargmann on the occasion of his 70th birthday.     

Morphometric analysis of the pineal complex of the golden hamster over a 24-hour light:dark cycle: II. The deep pineal in untreated and optically enucleated animals

The deep pineal gland of golden hamsters was morphometrically analyzed and quantitatively compared with the superficial pineal under a 14:10 lighting regime and following blinding. The deep pineal comprised 6-10% of the total pineal parenchymal tissue. Pinealocytes of the deep gland were smaller than the cells of the superficial pineal and showed a greater percent volume of Golgi bodies, rough endoplasmic reticulum, and dense-cored vesicles. Twenty-four-hour rhythms in nucleoli and Golgi bodies were found in deep pinealocytes. These rhythms were out of phase with comparable rhythms in the superficial pineal gland, suggesting that distinct subpopulations of pinealocytes are present within the respective parts. Blinding resulted in decreased nuclear and nucleolar volume, while the amount of smooth endoplasmic reticulum, Golgi bodies, dense bodies, and dense-cored vesicles increased significantly. Marginal increases were seen in mitochondria and lipid droplets. The greater abundance of those organelles involved in synthesis and secretion suggests enhanced cellular activity after blinding. Many of the morphological responses are similar to alterations in the pinealocytes of the superficial pineal following optic enucleation.     

Expression of synaptic vesicle trafficking proteins in the developing rat pineal gland

Adult mammalian pinealocytes contain several synaptic membrane proteins that are probably involved in the regulation of targeting and exocytosis of synaptic-like microvesicles (SLMVs). Immunohistochemical techniques have now demonstrated the spatiotemporal expression pattern of some of these proteins during rat pineal ontogenesis. Various synaptic vesicle trafficking proteins are detectable in proliferating epithelial cells of the pineal anlage even at embryonic day 17.5 (E 17.5), with the exception of syntaxin I (weakly expressed from E 19.5) and dynamin I (whose levels increase markedly during the first postnatal week). Numerous cells exhibiting strong immunoreactivity for synaptobrevin II, SNAP-25, synaptophysin, and munc-18-1 are distributed throughout the increasingly compact gland at E 19.5 and E 20.5; however, their number declines toward the proximal deep part of the organ. Groups of postmitotic cells situated at the surface of the developing gland exhibit marked immunoreactivity for the aforementioned proteins and lie close to the laminin-immunoreactive outer limiting basement membrane or to its remnants in regions of basement membrane dissolution. We also show that synthesis of vimentin and S-antigen seems to begin earlier during pineal development than previously recognized. Thus, synaptic vesicle trafficking proteins are the earliest molecular markers of pinealocyte differentiation known to date, being expressed well before the onset of rhythmic hormone secretion in the pineal gland, where they may play a role in morphogenetic events. Components of the extracellular matrix such as laminin may be critically involved in the upregulation of synaptic membrane protein expression. The dynamin immunostaining pattern indicates that SLMVs of pinealocytes begin to undergo regulated cycles of exo/endocytosis during postnatal week 1.     

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.     

The pineal organ of Raja clavata: Opsin immunoreactivity and ultrastructure

Summary The pineal organ of Raja clavata was studied by light and electron microscopy, including the immunocytochemical antiopsin reaction. The pineal organ of the ray consists of three portions: (i) a large proximal pineal, (ii) a long tube-like connecting stalk, and (iii) a short distal terminal enlargement. This latter end-vesicle lies in the deep connective tissue layers of the braincase. All portions of the pineal are composed of pinealocytes, intrinsic neurons, ependymal/glial cells, and bundles of nerve fibers embedded in thin neuropil formations. The inner segments of the pinealocytes protrude into the lumen in all parts of the organ and usually contain basal bodies and numerous mitochondria. Often, two outer segments were found to arise from the basal bodies of a single inner segment. By means of light-microscopic immunocytochemistry the outer segments showed a strong antiopsin reaction.The axons of the pinealocytes form ribbon-containing synapses on dendritelike profiles, which appear to belong to the intrinsic pineal neurons. There are other axo-dendritic synapses established by presynaptic terminals lacking ribbons and containing granular and synaptic vesicles. Pineal neurons may contain granular vesicles approximately 60–100 nm in diameter; their processes contribute to the bundles of unmyelinated axons.The fine structural organization of the pineal organ and the opsin immunoreactivity of the outer segments of the pinealocytes indicate a photoreceptive capacity of the organ. The double outer segments represent a peculiar multiplication of the photoreceptor structures.This investigation was supported by grants from the Deutsche Forschungsgemeinschaft to A. Oksche (Ok 1/24; 1/25: Mechanismen biologischer Uhren)On leave from the 2nd Department of Anatomy, Semmelweis OTE, Budapest, Hungary     

Morphologic evidence of photoreceptor differentiation of pinealocytes in the neonatal rat

The pineal body and the retina of the neonatal Sprague-Dawley rat were studied by light and electron microscopy, and the morphologic differentiation of the parenchymal cells of the pineal body was compared with that of the developing photoreceptor cells of the retina. Between the ages of 4 and 12 days after birth, some of the developing pinealocytes were observed to become elongated and polarized, with their nuclei located at one pole. "Synaptic" ribbons were observed within the cell body. At the opposite pole the cells developed elongated cell processes that initially contained microtubules and ribosomes. These cell processes projected into luminal spaces and were attached by structures resembling zonulae adherentes to the adjacent cells. Extending from the tips of the cell processes, cilia with a 9 + 0 arrangement were observed. Lamellated and vesicular membranes were noted at the tips of the cilia. Such morphologic differentiation, however, could be observed only in rats younger than 17 days. Comparison of the morphologic features of the neonatal pinealocytes with those of the developing retinal photoreceptor cells showed much similarity. It is suggested that the pinealocytes of the neonatal rat undergo "photoreceptor-like" differentiation during a transient neonatal period. Such morphologic differentiation may provide an explanation for light-induced biochemical changes described in neonatal rats whose eyes had been enucleated.     

Glutamate immunoreactivity is enriched over pinealocytes of the gerbil pineal gland

Summary Mammalian pinealocytes have been shown to contain synaptic-like microvesicles with putative secretory functions. As a first step to elucidate the possibility that pinealocyte microvesicles store messenger molecules, such as neuroactive amino acids, we have studied the distributional pattern of glutamate immunoreactivity in the pineal gland of the Mongolian gerbil (Meriones unguiculatus) at both light- and electron-microscopic levels. In semithin sections of plastic-embedded pineals, strong glutamate immunoreactivity could be detected in pinealocytes throughout the pineal gland. The density of glutamate immunolabeling in pinealocytes varied among individual cells and was mostly paralled by the density of immunostaining for synaptophysin, a major integral membrane protein of synaptic and synaptic-like vesicles. Postembedding immunogold staining of ultrathin pineal sections revealed that gold particles were enriched over pinealocytes. In particular, a high degree of immunoreactivity was associated with accumulations of microvesicles that filled dilated process terminals of pinealocytes. A positive correlation between the number of gold particles and the packing density of microvesicles was found in three out of four process terminals analyzed. However, the level of glutamate immunoreactivity in pinealocyte process endings was lower than in presumed glutamatergic nerve terminals of the cerebellum and posterior pituitary. The present results provide some evidence for a microvesicular compartmentation of glutamate in pinealocytes. Our findings thus lend support to the hypothesis that glutamate serves as an intrapineal signal molecule of physiological relevance to the neuroendocrine functions of the gland.     

The Pineal Gland of the Mink, Mustela vison: Light-, Fluorescence- and Electron Microscopical Studies

The pineal gland of normal and experimental female mink has been studied by light-, fluorescence- and electron microscopy. The general structure of the mink pineal is described. Two main cell types are recognized. One, termed pinealocyte, predominates in number. Though slight morphological differences (e.g. electron density of the cytoplasm and content of organelles) were observed, this study indicates that the pineal of mink only contains one single population of pinealocytes. The other, termed glial cell, inserted between the pinealocytes, is characterized by the presence of elongated processes, containing microfilaments. Different treatments (ovariectomy and LH—RH administration) and different endocrine states during the year induced morphological changes in the pinealocytes. A rich network of nerve fibres containing electron-dense granules (40–50 nm) is observed. Microspectrofluorometrically these fibres exhibit the spectral characteristics of cateholamines. All the pinealocytes show a yellow fluorescence. This cellular fluorophor shows the same microspectrofluorometric characteristics as does the fluorophor of serotonin. Occasionally, synaptic ribbons are observed in the perikaryon and the processes of the pinealocytes. A large number of cellular junctions between pinealocytes and endothelial cells is present. Their presumed function(s) are discussed. There is evidence of a blood-brain barrier within the mink pineal gland.     

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.     

The pineal gland of the gerbil,Meriones unguiculatus

Morphometric analytical procedures were employed to study the pineal gland of the Mongolian gerbil following superior cervical ganglionectomy (SCGX). The purpose of this study was to define the effects of sympathetic denervation on the morphology of the gland at two time periods, 0500 and 1900 h (one hour before lights-on and lights-off, respectively). Fluorescence histochemistry was employed to determine catecholamine and indoleamine content in intact and denervated pineal glands. After SCGX, the pinealocytes decrease in size, concretions are prevented from forming, and the yellow fluorescence in the gland is lost. Following denervation a depression in the volume of most of the pinealocyte organelles, i.e., SER, RER/ribosomes, free cytoplasm, mitochondria and presumptive secretory vesicles, was also observed. However, synaptic ribbons increased in volume in the gerbils that had been killed at 1900 h. It appears that the sympathetic innervation to the pineal gland is a requirement for the presumptive secretory activity of the pinealocytes.