Calcified inclusions in the superficial pineal gland of the mongolian gerbil, Meriones unguiculatus.

A histological and histochemical study of the pineal gland of neonatal, juvenile and adult gerbils is described. Calcified inclusions appear within pinealocytes in the superficial pineal about the third week of age, and the incidence of inclusions increased with age until, by the eleventh week, they are found in all animals. The inclusions contain an organic matrix composed of a carbohydrate, probably an acid mucopolysaccharide, complexed to protein. Calcification does not occur in the deep pineal. The data are interpreted to indicate that the formation of calcified inclusions is a normal process within the gerbil pineal. The similarity of the process of calcification in the gerbil and in the human pineal suggests that the gerbil may be an animal of choice for the controlled study of the phenomenon of pineal calcification.     

Fine-structural study of the pineal body of the monkey (Macaca fuscata) with special reference to synaptic formations

Summary Various types of synaptic formations on pinealocytes and pineal neurons were found in the pineal body of Macaca fuscata. Axo-somatic synapses of the Gray type-II category were detected on the pinealocyte cell body. Gap junctions and ribbon synapses were observed between adjacent pinealocytes. About 70 nerve-cell bodies were detected in one half of the whole pineal body bisected midsagittally. They were localized exclusively deep in the central part. When examined electron-microscopically, they were found to receive ribbon-synapse-like contacts from pinealocytic processes. They also received synaptic contacts of the Gray type-I category on their dendrites, and those of the Gray type-II category on their cell bodies from nerve terminals of unknown origin. All these synapse-forming axon terminals contained small clear vesicles. Thus, the pineal neurons of the monkey, at least in part, are suggested to be derived from the pineal ganglion cells in the lower vertebrates and not from the postganglionic parasympathetic neurons. The functional significance of these observations is discussed in relation to the innervation of the pineal body of the monkey.     

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.     

Ultrastructural visualization of exocytosis in the pig pineal gland

Summary The pinealocytes of the pig contain conspicuous dense bodies, the nature and role of which are not yet fully elucidated. The aim of this study was to demonstrate whether or not these structures are involved in the secretion process. The tannic acid-Ringer incubation (TARI)-method, which allows a clear-cut ultrastructural study of secretory discharge by exocytosis, has been used. The results indicate that pig pinealocytes release the content of the dense bodies with an amorphous inner structure into the extracellular space via exocytosis and that this secretion is quantitatively important. The secreted material is proteinaceous in nature; this indicates that polypeptides are released by the pineal.     

The pineal region in the opossum,Didelphis virginiana

Summary In the pineal region of the opossum, Didelphis virginiana, two types of cells predominate: 1) pinealocytes, and 2) fibrous astrocytes. Pinealocytes are characterized by the presence of prominent Golgi bodies, numerous clear and dense-cored vesicles, sensory cilia (9+0), vesicle-crowned rods, and condensation of a material that was always associated with the rough endoplasmic reticulum. In addition, two other cell types are occasionally seen. These include 1) neuron-like cells, and 2) darker staining cells of unknown identity. The endoplasmic reticulum of the darker staining cells is typically expanded and filled with an amorphous substance. Although the pineal region is small in size, the present findings suggest that pinealocytes in this species are metabolically active cells displaying a secretory function. Moreover, the presence of sensory cilia (9+0) and vesicle-crowned rods indicates that pinealocytes of the opossum are phylogenetically related to the photoreceptor cells found in the pineal organ of lower vertebrates.     

Pineal gland in rats with 7,12-dimethylbenz(a)anthracene-induced mammary tumors subjected to manipulations known as enhancers of pineal actions

The ultrastructure of pinealocytes was studied in rats with 7,12-dimethylbenz(a)anthracene-induced mammary tumors which were subjected to experimental manipulations known as enhancers of pineal actions (anosmia, underfeeding or cold exposure). In these animals we found: (I)--more nuclei with deep nuclear invaginations; (II)--a large number of cytoplasmic organelles, including lipid droplets, myeloid bodies, synaptic ribbons and lysosomes; (III)--numerous degenerative changes. In general, we found an increase in structural features related to pineal photoneuroendocrine activity. Our results indicate that pineal-dependent inhibition of neoplastic growth induced by these experimental manipulations, previously reported, can be mediated through an increase in pineal metabolic activity.     

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     

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.     

Inclusion bodies in pinealocytes of the cotton rat (Sigmodon hispidus)

Pinealocytes of the cotton rat (Sigmodon hispidus) often contain large (2-6 micron diameter) intracytoplasmic inclusions, the function of which is not known. These inclusions may represent nucleolus-like bodies, mineral deposits, secretory products or viral inclusions. In this study these inclusions were classified as type A, B or C inclusions based on the amount of electron-dense material interspersed within the finely granular material comprising the bulk of these inclusions. Each type of inclusion was analyzed by X-ray microanalysis and enzymatic proteinaceous digestion. X-ray microanalysis of these inclusions differed both quantitatively and semiquantitatively from that of human or gerbil pineal concretions, the latter two of which are extracellular deposits. Pronase, a proteolytic enzyme, digested the electron-dense material only after longer times of tissue exposure to this enzyme in contrast to the easily digested, finely granular matrix-like material of these inclusions. Such intrapinealocytic inclusions have only been observed in the cotton rat. Their functional significance remains unknown.     

Ultrastructural evidence for the presence of secretory cells in the pineal parenchyma ofHeteropneustes fossilis

There is an extensive literature dealing with the study of indoles, especially serotonin and melatonin, but with considerably less emphasis on the cells and cell types involved in the synthetic process. In the present electron microscopical investigation of the pineal end vesicle ofHeteropneustes fossilis, pinealocytes have been revealed in the pineal parenchyma characterized with extensive synthetic apparatus viz., rough endoplasmic reticulum, free ribosomes, lipid droplets, mitochondria and Golgi bodies. Two sub-populations of the pinealocytes are easily distinguishable on the basis of electron opacity and the preponderance of one or other morphological profile: light cells and dark cells. Light cells represent the active phase of secretion while dark cells represent the storage and release phase of secretion. A neuroendocrine role for the pineal body inHeteropneustes fossilis is suggested which may be significant in view of the nocturnal habit of the fish. Dedicated to my father, Prof. C B L Srivastava     

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     

Ultrastructural study of the distribution of calcium in the pineal gland of the rat subjected to manipulation of the photoperiod

Summary Using the pyroantimoniate technique, a study was conducted at electron microscope level on the distribution of the calcium ion in the pineal glands of normal adult Sprague-Dawley rats with initial weights of 150–200 g subjected to a 12:12 light dark cycle and others under the same conditions were subjected to modifications in the noradrenergic signal, such as continuous illumination over 7 days, blinding by bilateral enucleation (7 or 90 days) before sacrifice and bilateral superior cervical gangliectomy at 21 days before sacrifice. All the animals were sacrificed by decapitation, half of them at midday and the other half at midnight. Abundant fine precipitations of calcium were found in the intercellular spaces of the pineal glands of the normal rats. By contrast, in the gangliectomized animals subjected to constant illumination and chronic binding these precipitations were few in number. Additionally, two types of pinealocytes were observed regarding the distribution and concentration of intracytoplasmic calcium in both the normal and experimentally manipulated animals. Type I correspond to the classic light pinealocytes, with an absence of intracytoplasmic precipitations, although in the normal and gangliectomized animals sacrificed at midnight it was possible to observe fine deposits inside the mitochondrial matrix. Type II correspond to the classic dark pinealocytes, with a dense cytoplasmic matrix and numerous deposits of intracytoplasmic and intranuclear calcium; these were never seen in the type I 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.     

Immunocytochemical and electron-microscopic investigations of the pineal organ in adult agamid lizards,Uromastix hardwicki

Summary Lacertilian species display a remarkable diversity in the organization of the neural apparatus of their pineal organ (epiphysis cerebri). The occurrence of immunoreactive S-antigen and opsin was investigated in the retina and pineal organ of adult lizards, Uromastix hardwicki. In this species, numerous retinal photoreceptors displayed S-antigen-like immunoreactivity, whereas only very few pinealocytes were labeled. Immunoreactive opsin was found neither in retinal photoreceptors nor in pinealocytes. Electron microscopy showed that all pinealocytes of Uromastix hardwicki resemble modified pineal photoreceptors. A peculiar observation is the existence of a previously undescribed membrane system in the inner segments of these cells. It is evidently derived from the rough endoplasmic reticulum but consists of smooth membranes. The modified pineal photoreceptor cells of Uromastix hardwicki were never seen to establish synaptic contacts with somata or dendrites of intrapineal neurons, which are extremely rare. Vesiclecrowned ribbons are prominent in the basal processes of the receptor cells, facing the basal lamina or establishing receptor-receptor and receptor-interstitial type synaptoid contacts. Dense-core granules (60–250 nm in diameter) speak in favor of a secretory activity of the pinealocytes. Attention is drawn to the existence of receptor-receptor and receptor-interstitial cell contacts indicating intramural cellular relationships that deserve further study.Supported by the Deutsche Forschungsgemeinschaft (Ko 758/31) and the Deutscher Akademischer Austauschdienst (Senior DAAD Research Fellowship to M.A.H.)     

Characteristic pattern of monoaminergic nerve fibers in the pineal organ of the monkey,Macaca fuscata

Summary Monoaminergic nerve fibers were studied in the pineal organ of the monkey, Macaca fuscata, by use of fluorescence and immunohistochemical procedures. Abundant formations of noradrenergic nerve fibers were observed in the pineal organ. They entered the parenchyma in the form of several coarse bundles via the capsule in the distal portion of the organ and spread throughout the organ after branching into smaller units. The density of the autonomic innervation decreased gradually toward the proximal portion of the organ. In the distal portion, numerous nerve fibers formed perivascular plexuses around the blood vessels and some fibers ran as bundles unrelated to the blood vessels in the stroma. Fine varicose fibers and bundles derived from these plexuses penetrated among the pinealocytes. However, only a few intraparenchymal fluorescent fibers were detected in the proximal third of the gland. With the use of serotonin antiserum serotonin-immunoreactive nerve fibers were clearly restricted to the ventroproximal part of the pineal organ. Although the somata of the pinealocytes showed intense immunoreactivity, their processes were not stained. In one exceptional case, clusters of pinealocytes displaying very intense immunoreactivity were found in an area extending from the distal margin of the ventral portion of the pineal stalk to the proximal portion of the pineal organ proper; these cells were bipolar or multipolar and endowed with well-stained processes.     

Monoamines and acetyl-cholinesterase in the pineal gland and habenula of the ferret

Summary A histochemical method for demonstrating amines by fluorescence showed that the pinealocytes of the ferret contained a high concentration of a yellow fluorophore (probably 5-HT). Numerous green-fluorescent (noradrenaline-containing) nerve fibres occurred around intrapineal blood vessels, between pinealocytes and in the N. conarii (which entered the gland caudally). A collection of neuron-like cells (the pineal ganglion) lay, surrounded by a meshwork of nerve fibres, in the posterior part of the pineal. Neither the cells nor the fibres of the pineal ganglion contained monoamines, but both showed the presence of acetyl-cholinesterase which otherwise was found in the pineal only in fibres which stretched from the ganglion towards the cranial pole of the gland. The medial habenular nucleus showed a remarkable perivascular green fluorescence not seen in the lateral habenular nucleus nor anywhere else in the adjacent diencephalon and brain stem. The cells and fibres of this nucleus also contained much acetyl-cholinesterase.Bilateral superior cervical ganglionectomy, or treating animals with reserpine, removed the green fluorescence from both pineal nerve fibres and the habenula. Ganglionectomy also resulted in a progressive alteration in the colour of the parenchymal fluorescence from yellow to green; the original yellow colour was restored by treating ganglionectomised animals with nialamide (a monoamine oxidase inhibitor). L-Dopa, 5-hydroxytryptophan or nialamide, alone or in combination, had no effect on the fluorescence of the nerve fibres or cells of the pineal, or on the habenula.These results are related to previous findings that pinealectomy or ganglionectomy prevents the acceleration by artificial light of oestrus in ferrets.     

Ultrastructural study of the distribution of calcium in the pineal gland of the rat subjected to manipulation of the photoperiod

Using the pyroantimoniate technique, a study was conducted at electron microscope level on the distribution of the calcium ion in the pineal glands of normal adult Sprague-Dawley rats with initial weights of 150-200 g subjected to a 12:12 light dark cycle and others under the same conditions were subjected to modifications in the noradrenergic signal, such as continuous illumination over 7 days, blinding by bilateral enucleation (7 or 90 days) before sacrifice and bilateral superior cervical gangliectomy at 21 days before sacrifice. All the animals were sacrificed by decapitation, half of them at midday and the other half at midnight. Abundant fine precipitations of calcium were found in the intercellular spaces of the pineal glands of the normal rats. By contrast, in the gangliectomized animals subjected to constant illumination and chronic binding these precipitations were few in number. Additionally, two types of pinealocytes were observed regarding the distribution and concentration of intracytoplasmic calcium in both the normal and experimentally manipulated animals. Type I correspond to the classic light pinealocytes, with an absence of intracytoplasmic precipitations, although in the normal and gangliectomized animals sacrificed at midnight it was possible to observe fine deposits inside the mitochondrial matrix. Type II correspond to the classic dark pinealocytes, with a dense cytoplasmic matrix and numerous deposits of intracytoplasmic and intranuclear calcium; these were never seen in the type I pinealocytes.     

Peculiar behavior of the nucleolus and appearance of cytoplasmic nucleolus-like bodies in the roottip meristems ofBrodiaea uniflora Engl. Grown at low temperature

Summary Cytoplasmic nucleolus-like bodies, which showed vast variation both in size and in number per cell, were sometimes found in the telophase cells ofBrodiaea uniflora. When the plants were grown at low temperature, the frequency of telophase cells bearing cytoplasmic nucleolus-like bodies increased with the lapse of days. In contrast, growth at moderate temperature reduced this frequency. Prolonged exposure of the plants to low temperature caused peculiar phenomena concerning the behavior of the nucleolus and nucleolar material: 1. retention of nucleolar remnants at high frequency in metaphase, 2. pulverization of the nucleolar remnants into a great number of minute, fluffed fragments during metaphase, 3. appearance of dot-like nucleolar material in anaphase, and 4. appearance of nucleolus-like bodies, sometimes more than 10 m in diameter, in telophase. All these structures were strongly impregnated with silver. Electron microscopy revealed that both the nucleolar remnant and the nucleolus-like body consisted primarily of fibrils. Our observations clearly demonstrate that the nucleolus-like bodies are derived from the fibrillar component of the nucleolus and are formed by fusion of dot-like nucleolar material during anaphase.     

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.     

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.