Types Of Pinealocytes Research Articles

RNA Sequencing of Single Pineal Cells.

The pineal gland presents a powerful genetic tool to study a broad range of physiological processes. It has been instrumental as a model in understanding transduction processes and daily changes in gene expression and holds great promise in understanding development. Currently, the field is at an exciting point, with methods available for the isolation of individual cells and, as presented here, the preparation of these single cells for sequencing. The resulting cellular transcriptomes have played a role in categorizing cells in the pineal gland, with current estimates including two types of pinealocytes, three types of astrocytes, two types of microglia, and two types of endothelial cells, including the poorly understood vascular and meningeal cell. The methods described in this chapter will serve to support and advance cellular studies of the pineal gland in the twenty-first century.

The pineal gland of the shrew (Blarina brevicauda and Blarina carolinensis): a light and electron microscopic study of pinealocytes

The pineal gland structure and ultrastructure in the Northern (Blarina brevicauda) and Southern short-tailed shrew (Blarina carolinensis) are described by light and electron microscopy. Results observed were similar to other mammals of Insectivora described previously, specifically, the hedgehog (Erinaceus europaeus) and the Old World mole (Talpa europea). Two different types of pinealocytes were noticed by electron microscopy, in addition to relatively few glial cells. Granular vesicles were not noticed in abundance. The granular endoplasmic reticulum was observed and studded with vesicles. The golgi apparatus was well developed and appeared often. Synaptic ribbons were observed in several different formations consisting of ribbons and/or rods. The ciliary derivative, the rudimentary photoreceptor structures found in the pinealocytes of population I, was noticed in a 9 + 0 tubular pattern. Within these semifossorial shrews, the relationship between specific intracellular organelles and their function was discussed.

CELLULAR CONSTITUENTS OF THE PINEAL PARENCHYMA IN THE ALBINO RAT: EFFECT OF DIFFERENT PHOTOPERIODS

The pineal gland is an endocrine organ synthesizing and secreting substances of well-defined hormonal nature. The pinealocytes represent the major cellular component of its parenchyma (Vollrath, 1981). The term pinealocytes is used as an inclusive term i.e. all pineal cells excluding neurons and glial elements. Several attempts have been made to classify the pinealocytes into structurally and functionally distinct groups of cells.Blumfield and Tapp (1970) studying the rat pineal have distinguished three types of pinealocytes according to the nuclear size and suggested a correlation with pinealocyte function. Differences in mitochondria have also been recorded in hamsters where the pinealocytes were classified into P1 pinealocytes with small cristiforrn mitochondria and P2 pinealocytes with mitochondria exhibiting a plexiform array of crests in a dense matrix (Clabough, 1971). Pevet and Collin (1976) studying the mole pineals have introduced another classification according to the number and form ofpinealocyte processes. Using the terminology employed to nerve cells, they have distinguished three types of pinealocytes. Type I pinealocytes with two processes have been considered bipolar cells, type II pinealocytes withone process have been classified as unipolar elements and type III pinealocytes have been regarded as multipolar cells due to the presence of multiple processes. Light and dark pinealocytes have also been distinguished in many species, e.g. mouse (Upson et aI., 1976), rat (Johnson, 1980; MiJin et aI., 1990; Humbert and Pevet, 1995), horse (Cozzi, 1986), rabbit (Krakowski and Cieciura 1992), gerbil (Redecker, 1993) and pig (Przybylska, 1989; Lewczuk et al., 1994). However, the nature of the two forms of cells was controversial. Some investigators have considered them as two distinct cell populations (Upson et al., 1976; MiJin et aI., 1990). Others have regarded them as different functional states of one and the same cell type (Przybylska, 1989; Redecker, 1993).

The pineal organ as a folded retina: Immunocytochemical localization of opsins

The most simple pineal complex (the pineal and parapineal organs of lampreys), consists of saccular evaginations of the diencephalic roof, and has a retina-like structure containing photoreceptor cells and secondary neurons. In more differentiated vertebrates, the successive folding of the pineal wall multiplies the cells and reduces the lumen of the organ, but the pattern of the histological organization remains similar to that of lampreys; therefore, we consider the histological structure of the pineal organ of higher vertebrates as a ‘folded retina’. The cell membrane of several pineal photoreceptor outer-segments of vertebrates immunoreact with anti-retinal opsin antibodies supporting the view of retina-like organization of the pineal. Some other pineal outer segments do not react with retinal anti-opsin antibodies, a result suggesting the presence of special pineal photopigments in different types of pinealocytes that obviously developed during evolution. The chicken pinopsin, detected in the last years, may represent one of these unknown photopigments. Using antibodies against chicken pinopsin, we compared the immunoreactivity of different photoreceptors of the pineal organs from cyclostomes to birds at the light and electron microscopic levels. We found pinopsin immunoreaction on all pinealocytes of birds and on the rhodopsin-negative large reptilian pinealocytes. As the pinopsin has an absorption maximum at 470 nm, these avian and reptilian immunoreactive pinealocytes can be regarded as green-blue light-sensitive photoreceptors. Only a weak immunoreaction was observed on the frog and fish pinealocytes and no reaction was seen in cyclostomes and in the frontal organ of reptiles. Some photoreceptors of the retina of various species also reacted the pinopsin antibodies, therefore, pinopsin must have certain sequential similarity to individual retinal opsins of some vertebrates.

The pineal organ as a folded retina: Immunocytochemical localization of opsins*

The most simple pineal complex (the pineal and parapineal organs of lampreys), consists of saccular evaginations of the diencephalic roof, and has a retina‐like structure containing photoreceptor cells and secondary neurons. In more differentiated vertebrates, the successive folding of the pineal wall multiplies the cells and reduces the lumen of the organ, but the pattern of the histological organization remains similar to that of lampreys; therefore, we consider the histological structure of the pineal organ of higher vertebrates as a ‘folded retina’. The cell membrane of several pineal photoreceptor outer‐segments of vertebrates immunoreact with anti‐retinal opsin antibodies supporting the view of retina‐like organization of the pineal. Some other pineal outer segments do not react with retinal anti‐opsin antibodies, a result suggesting the presence of special pineal photopigments in different types of pinealocytes that obviously developed during evolution. The chicken pinopsin, detected in the last years, may represent one of these unknown photopigments. Using antibodies against chicken pinopsin, we compared the immunoreactivity of different photoreceptors of the pineal organs from cyclostomes to birds at the light and electron microscopic levels. We found pinopsin immunoreaction on all pinealocytes of birds and on the rhodopsin‐negative large reptilian pinealocytes. As the pinopsin has an absorption maximum at 470 nm, these avian and reptilian immunoreactive pinealocytes can be regarded as green‐blue light‐sensitive photoreceptors. Only a weak immunoreaction was observed on the frog and fish pinealocytes and no reaction was seen in cyclostomes and in the frontal organ of reptiles. Some photoreceptors of the retina of various species also reacted the pinopsin antibodies, therefore, pinopsin must have certain sequential similarity to individual retinal opsins of some vertebrates.

The pineal organ, its hormone melatonin, and the photoneuroendocrine system.

The vertebrate pineal organ rhythmically synthesizes and secretes melatonin during nighttime and forms an essential component of the photoneuroendocrine system which allows humans and animals to measure and keep the time. Regulation of the melatonin biosynthesis depends on signals from photoreceptors perceiving and transmitting environmental light stimuli and endogenous oscillators generating a circadian rhythm which is independent from any environmental time cue (zeitgeber). In nonmammalian species the photoreceptors responsible for regulating melatonin biosynthesis reside within the pineal organ itself. In several nonmammalian species (e.g., lamprey, zebra fish, house sparrow, chicken) the pineal organ is also capable of generating circadian rhythms and thus serves all key functions of the photoneuroendocrine system: photoreception, endogenous rhythm generation, and production of neurohormones. These may even be accomplished by a single "photoneuroendocrine" cell. In mammals the pineal organ has lost both the direct light sensitivity and the capacity of generating circadian rhythms, and melatonin biosynthesis is regulated by retinal photoreceptors and a circadian oscillator located in the suprachiasmatic nucleus of the hypothalamus. Due to this spatial separation the photoneuroendocrine system of mammals comprises neuronal and neuroendocrine pathways which interconnect its components. The neuronal pathways involve circuits of both the central and the peripheral nervous systems, and as an important final link noradrenergic sympathetic nerve fibers. The suprachiasmatic nucleus appears as a major target of melatonin in mammals. The pineal hormone may thus be involved in a feedback loop of the mammalian photoneuroendocrine system. The present comparative contribution considers, after a short survey of classical findings on the phylogenetic development and the gross anatomy of the pineal complex, cytoevolutionary and cell biological aspects of the various types of pinealocytes as well as the afferent and efferent innervation of the pineal organ (pinealofugal and pinealopetal neuronal pathways). Moreover, emphasis is placed on receptor mechanisms, second messenger systems (Ca2+ and cyclic AMP), transcription factors (e.g, CREB and ICER), and their roles for regulation of melatonin biosynthesis. Finally, the action, targets, and receptors of melatonin are dealt with. The synoptic approach of this contribution, which combines anatomical and ultrastructural findings with cell and molecular biological results, confirms the functional significance of the melatonin-synthesizing pineal organ as an important component of the photoneuroendocrine system and stresses the importance of this organ as a model to study signal transduction mechanisms both in photoreceptors and in neuroendocrine cells.

Dense accumulations of synaptic-like microvesicles in 'dark' pinealocytes of the gerbil pineal gland.

In an electron-microscopical study the occurrence and ultrastructural features of electron-dense 'dark' variants of pinealocytes were evaluated in the gerbil pineal gland. A few 'dark' pinealocytes, which tended to form small clusters of contiguous cells, could consistently be detected in pineals fixed and embedded by various procedures. Apart from the different degree of electron density, the only conspicuous difference between 'dark' and electron-lucent 'light' pinealocytes concerned their compartment of synaptic-like microvesicles. Thus, both variants of pinealocytes contained abundant clear microvesicles of variable size which accumulated in dilated process terminals. However, the vesicles within the process endings of 'dark' pinealocytes showed an unusually dense arrangement throughout the cytoplasm. As was demonstrated by immunogold staining, the accumulations of vesicles in the 'dark' terminals contained synaptophysin, a major synaptic vesicle-associated protein. This protein is present in small clear vesicles with putative secretory functions in a wide variety of neuroendocrine cells and has previously been shown to be a common constituent of microvesicles in mammalian pinealocytes. Since gerbil pinealocytes displayed distinct gradations of electron density, their ultrastructural heterogeneity may be the expression of different states of secretory activity of one pinealocyte cell type. On the other hand, differences in the content of synaptic-like microvesicles in the process terminals of 'light' and 'dark' cells could also indicate a principal functional heterogeneity of the microvesicular compartment among pinealocytes, pointing to the existence of different types of pinealocytes.

Tissue kallikrein in the rat pineal gland: An immunocytochemical study

Tissue kallikrein in the rat pineal gland was immunocytochemically investigated with the aid of specific antiserum against rat urinary kallikrein. We also compared the tissue kallikrein immunoreactivity of the pineal gland with that of the submandibular gland and kidney, which have been well established as tissue kallikrein-synthesizing organs. The cytoplasm of pinealocytes from both the superficial and the deep portion of the gland exhibited specific immunolabeling for tissue kallikrein, but the immunoreaction was weaker than that observed in exocrine organs. Two types of tissue kallikrein-immunoreactive pinealocytes were distinguished; the first predominant type displayed moderate immunostaining, whereas a small number of cells, the second type, were so intensely labeled that their cytoplasmic processes were clearly outlined. The results of the present study suggest the existence of different types of pinealocytes and a potential physiological role of tissue kallikrein in the rat pineal gland.

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.

Pinealocytes immunoreactive with antisera against secretory glycoproteins of the subcommissural organ: A comparative study

By means of light-microscopic immunocytochemistry two polyclonal antibodies (AFRU, ASO; see p. 470) directed against secretory glycoproteins of the subcommissural organ were shown to cross-react with cells in the pineal organ of lamprey larvae, coho salmon, a toad, two species of lizards, domestic fowl, albino rat and bovine (taxonomic details, see below). The AFRU-immunoreactive cells were identified as pinealocytes of the receptor line (pineal photoreceptors, modified photoreceptors or classical pinealocytes, respectively) either due to their characteristic structural features or by combining AFRU-immunoreaction with S-antigen and opsin immunocytochemistry in the same or adjacent sections. Depending on the species, AFRU- or ASO-immunoreactions were found in the entire perikaryon, inner segments, perinuclear area, and in basal processes facing capillaries or the basal lamina. In most cases, only certain populations of pinealocytes were immunolabeled; these cells were arranged in a peculiar topographical pattern. In lamprey larvae, immunoreactive pinealocytes were observed only in the pineal organ, but not in the parapineal organ. In coho salmon, the immunoreaction occurred in S-antigen-positive pinealocytes of the pineal end-vesicle, but was absent from S-antigen-immunoreactive pinealocytes of the stalk region. In the rat, AFRU-immunoreaction was restricted to S-antigen-immunoreactive pinealocytes found in the deep portion of the pineal organ and the habenular region. These findings support the concept that several types of pinealocytes exist, which differ in their molecular, biochemical and functional features. They also indicate the possibility that the AFRU- and ASO-immunoreactive material found in certain pinealocytes might represent a proteinaceous or peptide compound, which is synthesized and released from a specialized type of pinealocyte in a hormone-like fashion. This cell type may share functional characteristics with peptidergic neurons or paraneurons.

Evidence for the presence of two 24-h rhythms 180 degrees out of phase in the pineal gland of male Pirbright-White guinea pigs as monitored by counting "synaptic" ribbons and spherules.

Previous studies have shown that the "synaptic" bodies of mammalian pinealocytes are a heterogeneous group of organelles. Whereas "synaptic" ribbons (SR) exhibit a day/night rhythm with small numbers during daytime and high numbers at night, the so-called "synaptic" spherules (SS) show species-specific differences in their rhythmicity. In the present study the numbers of SR and SS were monitored in male guinea pigs over a full 24-h period at 4-hourly intervals (LD 12:12, lights on at 7:00). The results obtained show that the rhythms of SR numbers and SS numbers are out of phase by 180 degrees. SR numbers are small during daytime and high at night, whereas SS numbers are high during daytime and low at night. As there are no indications that SR are transformed into SS and vice versa and as SR and SS lie in different parenchymal areas it is suggested that they characterize different types of pinealocytes with differing rhythmicities. - Serum melatonin levels were low during daytime (30 pg/ml) and increased at night to reach a peak (84 pg/ml) at 24:00 h.

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.

The pineal gland of the mole (Talpa europaea L.).

The ultrastructure of the pinealocytes in the fetal mole (Talpa europaea) was examined and compared with that in the adult mole. The parenchyma of the fetal pineal gland consists primarily of pinealocytes with very few dispersed "glial" cells. Three different morphological types of pinealocytes (I, II and III) were observed. Pinealocytes of types I and II, homologous to the rudimentary photoreceptor cells of lacertilians and birds, were commonly found, especially around the pineal lumen, which is still present in the fetus. These results support the concept of the phylogenetic origin of mammalian pinealocytes from the pineal photoreceptor cells of the nonmammalian vertebrates. Considering their synthetic/secretory activity, the pinealocytes of the mole fetus are characterized by the presence of accumulations of proteinaceous material (APM) in the cisternae of the granular endoplasmic reticulum (GER). Two types of APM were also found. One type shows a paracrystalline organization as described in the adult mole. In the fetus, however, the paracrystalline-organized APM occur only infrequently. A progressive transformation, via an increase in size or fusion of vacuoles containing material originating from the cisternae of the GER in APM, free of a paracrystalline structure, has been observed. Granular vesicles (GV) originating from the Golgi saccules were rarely observed. Sometimes GV and APM were found to be present in the same cell.

Les pinéalocytes de mammifère: diversité, homologies, origine. Etude chez la taupe adulte ( Talpa europaea L.)

en An electron microscopic investigation of the pineal organ of an adult mammal (the mole Talpa europaa L.) revealed the presence of several types of pinealocytes sensu stricto. In comparing three of these with one another, on the one hand, and with typical and rudimentary photoreceptor cells, characteristic of the epiphysis cerebri of non-mammalian vertebrates, on the other hand, the following homologies can be drawn at the cellular level: homology between a cilium (9 + 0 pattern) and the outer segment, homology (shape, structure and polarity of the organelles) between a process of large diameter and the inner segment, homology between the cell bodies and other processes, and homologies between the vesicle-crowned lamellae and the synaptic ribbons. The presence of three principal types of pinealocytes and of intermediary types can be explained as the result of different stages of cell differentiation during ontogenesis, which are maintained in the adult. The ultrastructural homologies now established, suggest that ancestral reptilian photoreceptor cells are the forerunners of mammalian pinealocytes. Our results, in combination with those recently obtained by other authors studying pineal development, support the existence of a sensory cell line in the vertebrate pineal organ.