Pineal Cells Dissociation and Culture: Isolated Pinealocytes, Isolated Astrocytes, and Co-culture.

Mammalian pineal glands are composed mostly of pinealocytes, which are the melatonin secretory cells, and also importantly of glial cells in special astrocytes. With the aim of studying the interactions between pinealocytes and astrocytes, the methodologies for obtaining and maintaining isolated pinealocytes and astrocytes in culture were standardized, in addition to the co-culture of both cell types. Some works of our group were published on the interactions between isolated astrocytes and pinealocytes from the pineal gland of Wistar rats, considering the modulatory role of glutamate and angiotensin on the synthesis of melatonin. In this chapter, the methodologies for obtaining and maintaining astrocytes and pinealocytes culture as well as co-culture of these two cell types will be presented.

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.

Investigation of the human pineal gland 3D organization by X-ray phase contrast tomography.

Pineal gland (PG) is a part of the human brain epithalamus that plays an important role in sleep, circadian rhythm, immunity, and reproduction. The calcium deposits and lesions in PG interfere with normal function of the organ and can be associated with different health disorders including serious neurological diseases. At the moment, the detailed mechanisms of PG calcifications and PG lesions formation as well as their involvement in pathological processes are not fully understood. The deep and comprehensive study of the structure of the uncut human PG with histological details, poses a stiff challenge to most imaging techniques, due to low spatial resolution, low visibility or to exceedingly aggressive sample preparation. Here, we investigate the whole uncut and unstained human post-mortem PGs by X-ray phase contrast tomography (XPCT). XPCT is an advanced 3D imaging technique, that permits to study of both soft and calcified tissue of a sample at different scales: from the whole organ to cell structure. In our research we simultaneously resolved 3D structure of parenchyma, vascular network and calcifications. Moreover, we distinguished structural details of intact and degenerated PG tissue. We discriminated calcifications with different structure, pinealocytes nuclei and the glial cells processes. All results were validated by histology. Our research clear demonstrated that XPCT is a potential tool for the high resolution 3D imaging of PG morphological features. This technique opens a new perspective to investigate PG dysfunction and understand the mechanisms of onset and progression of diseases involving the pineal gland.

Fluoride-Free Diet Stimulates Pineal Growth in Aged Male Rats.

The pineal gland is a naturally calcifying endocrine organ which secretes the sleep-promoting hormone melatonin. Age-related changes of the pineal have been observed, including decreased pinealocyte numbers, increased calcification, and a reduction in melatonin production. Since fluoride is attracted to calcium within the pineal gland, this study sought to examine the effects of a fluoride-free diet on the morphology of the pineal gland of aged male rats (26 months old). All animals had previously been raised on standard fluoridated food and drinking water. These control animals were compared to other animals that were placed on a fluoride-free diet ("fluoride flush") for 4 or 8 weeks. At 4 weeks, pineal glands from fluoride-free animals showed a 96% increase in supporting cell numbers and at 8 weeks a 73% increase in the number of pinealocytes compared to control animals. In contrast, the number of pinealocytes and supporting cells in animals given an initial 4-week fluoride flush followed by a return to fluoridated drinking water (1.2 ppm NaF) for 4 weeks were not different from control animals. Our findings therefore demonstrate that a fluoride-free diet encouraged pinealocyte proliferation and pineal gland growth in aged animals and fluoride treatment inhibited gland growth. These findings suggest that dietary fluoride may be detrimental to the pineal gland.

Developmental morphology of the turkey pineal organ. Immunocytochemical and ultrastructural studies.

Our previous study showed that the turkey pineal organ, in contrast to that of the chicken, is characterized by a follicular structure throughout the entire period of post-hatching life. Despite the preservation of the follicular organization, the histological structure of the pineal follicles in turkeys changes prominently with age. The present research was performed to investigate the cellular composition and organization of the follicle wall as well as the ultrastructure of parenchymal cells in the turkey pineal organ during the period of post-hatching development. Pineal organs were collected from female turkeys at 2 days, 2 weeks, 4 weeks, 10 weeks, 20 weeks, 30 weeks, 40 weeks, and 56 weeks post-hatching. The organs were prepared for immunocytochemical studies using antibodies against N-acetylserotonin O-methyltransferase (ASMT), glial fibrillary acidic protein (GFAP) and proliferating cell nuclear antigen (PCNA) and for ultrastructural examination. The results showed that regardless of age, the pineal follicle was formed by ASMT-immunopositive cells, among which rudimentary photoreceptor and secretory pinealocytes were identified. The second component of the follicle wall consisted of GFAP-immunopositive cells, as represented by ependymal-like and astrocyte-like cells. Rudimentary photoreceptor pinealocytes and ependymal-like cells formed the inner part of the follicle wall, while secretory pinealocytes and astrocyte-like cells created the outer part. Three forms of the pineal follicle structure characteristic of young (two days to ten weeks), young adult (20-30 weeks) and adult (40-56 weeks) turkeys were distinguished. These forms primarily differed in the relative dimensions of the inner and outer parts of the follicle wall. Ultrastructural studies showed prominent changes in the organization of rudimentary receptor pinealocytes during the investigated period of life. These cells developed until the age of 20 weeks, at which time they appeared as strongly elongated cells with a stratified, highly regular distribution of organelles. In adult turkeys, rudimentary receptor pinealocytes showed pronounced regressive changes; however, we never observed their transformation into cells of the secretory type. Secretory pinealocytes increased in number and size during the post-hatching period, which was especially pronounced after 20 weeks of age. The most prominent changes in the supporting cells included the intensification of GFAP-immunoreactivity due to the accumulation of filaments in the cytoplasm and the development of astrocyte-like cells. The increase in the number of secretory pinealocytes and astrocyte-like supporting cells resulted in the formation of two distinct parts of the follicle wall in the pineal organs of young adult and adult turkeys.

[Pharmacological correction of alterations of apoptosis of the neurons of the hypothalamus suprachiasmatic nucleus and pinealocytes during aging and stress.]

As is known, the pineal gland plays an important role in adaptogenesis, and the hypothalamus is one of the main links of the stress-reactive system and is involved in the regulation of the involution of the whole organism. So, the study of changes in these organs during stress and aging is very interesting. The aim of the work is to study the mechanisms of apoptosis of pinealocytes and neurosecretory cells of the suprachiasmatic nucleus of the hypothalamus during aging, stress, and under the conditions of pharmacological correction of involutional processes and stress response (antioxidant alpha-tocopherol acetate, immunomodulator cycloferon). We used Wistar rats as model, young (2-4 months) and old (30 months). Age-related features of the apoptosis dynamics of pinealocytes and neurosecretory cells of the hypothalamic suprachiasmatic nucleus were studied using TUNEL and immunohistochemistry, and the possibilities of pharmacological correction of apoptotic processes are determined. An age-dependent increase of apoptosis level of cells of suprachiasmatic nucleus and epiphysis in rats was revealed. The stress effect (immobilization) led to the intensification of cell death, more significant in older animals. The pineal gland and suprachiasmatic nucleus, traditionally regarded as regulators of circadian rhythms, are at the same time actively involved in general adaptation processes. The studied drugs (α-tocopherol-acetate, cycloferon, and their combination) have a pronounced anti-apoptotic, cytoprotective effect under physiological conditions during aging, as well as during non-specific emotional stress (immobilization) in young and old animals. The regulatory effect is accomplished by activating the expression of the anti-apoptotic protein Bcl-2 in the neurosecretory cells of the suprachiasmatic nucleus and pinealocytes.

Nonproliferative and Proliferative Lesions of the Rat and Mouse Endocrine System.

The INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) Project (www.toxpath.org/inhand.asp) is a joint initiative among the Societies of Toxicological Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP) and North America (STP) to develop an internationally accepted nomenclature for proliferative and nonproliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature for classifying microscopic lesions observed in the endocrine organs (pituitary gland, pineal gland, thyroid gland, parathyroid glands, adrenal glands and pancreatic islets) of laboratory rats and mice, with color photomicrographs illustrating examples of the lesions. The standardized nomenclature presented in this document is also available electronically on the internet (http://www.goreni.org/). Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous and aging lesions as well as lesions induced by exposure to test materials. A widely accepted and utilized international harmonization of nomenclature for endocrine lesions in laboratory animals will decrease confusion among regulatory and scientific research organizations in different countries and provide a common language to increase and enrich international exchanges of information among toxicologists and pathologists.

Adrenergic regulation of cytoplasmic structures related to secretory processes in pig pinealocytes-an ultrastructural, quantitative study.

Two structures, considered as secretory in nature, are present in the pinealocytes in of the domestic pig show the presence of two structures, which are considered as secretory in nature - the dense core vesicles (DCV) and the membrane bounded (dense) bodies (MBB). The latter are extremely numerous in pig pinealocytes (they occupy 6-20% of the cytoplasm), and the number of MBB changes under different physiological and experimental conditions. Norepinephrine is the main neurotransmitter that regulates the secretion of pineal melatonin. The present study was carried out to 1) clarify whether the DCV and their source - the Golgi apparatus (GA) - as well as the MBB are controlled by norepinephrine, 2) determine the effect of adrenergic stimulation on these structures, and 3) identify the receptors involved in the regulation of these structures. The studies were performed using a static organ culture of pig pineal explants. The explants were incubated in a control medium between 08:00 and 20:00 and in a medium with 10µM norepinephrine or alpha- or beta-adrenoceptor agonists between 20:00 and 08:00 on five consecutive days. The tissues were subsequently prepared for ultrastructural analysis. The results distinctly showed that the DCV, GA and MBB in pig pinealocytes are under adrenergic control. The stimulation of the beta-adrenoceptors resulted in an increase in the numerical density of the DCV and a decrease in the relative volume of the GA in the perikarya, while the incubation with agonists of the alpha1-adrenoceptors was ineffective. The relative volume of the MBB in the perikarya significantly decreased after treatment with both beta-agonists and alpha1-agonists, which suggested the involvement of two types of adrenoceptors in the regulation of these structures.

Immunofluorescence reveals unusual patterns of labelling for connexin43 localized to calbindin-D28K-positive interstitial cells in the pineal gland.

Gap junctions between cells in the pineal gland have been described ultrastructurally, but their connexin constituents have not been fully characterized. We used immunofluorescence in combination with markers of pineal cells to document the cellular localization of connexin43 (Cx43). Immunofluorescence labelling of Cx43 with several different antibodies was widely distributed throughout the pineal, whereas another connexin examined, connexin26, was not found in pineal but only in surrounding leptomeninges. Labelling apparently associated with plasma membranes was visualized either as fine Cx43-puncta (1-2 µm) or as unusually large pools of Cx43 ranging up to 4-7 µm in diameter or length. These puncta and pools were highly concentrated in perivascular spaces, where they were associated with numerous cells devoid of labelling for markers of pinealocytes (e.g. tryptophan hydroxylase and serotonin), and where they were minimally associated with blood vessels and lacked association with resident macrophages. Astrocytes labelled for glial fibrillary acidic protein were largely restricted to the anterior pole of the pineal gland, where they displayed only fine and sparse Cx43-puncta along their processes. Labelling for Cx43 was localized largely though not exclusively to the somata and long processes of a subpopulation of perivascular interstitial cells that were immunopositive for calbindin-D28K. These cells were often located among dense bundles or termination areas of sympathetic fibres labelled for tyrosine hydroxylase or serotonin. The results indicate that interstitial cells form abundant gap junctions composed of Cx43, and suggest that gap junction-mediated intracellular communication by these cells supports the activities of pinealocytes.

The pineal complex: a morphological and immunohistochemical comparison between a tropical (Paracheirodon axelrodi) and a subtropical (Aphyocharax anisitsi) characid species.

Cardinal neon Paracheirodon axelrodi and bloodfin tetra Aphyocharax anisitsi are two species of characids with high trade value as ornamental fish in South America. Although both species inhabit middle water layers, cardinal neon exhibits a tropical distribution and bloodfin tetra a subtropical one. In this work, we carried out an anatomical, histological and immunohistochemical study of the pineal complex of P. axelrodi and A. anisitsi. In both species, the pineal complex consisted of three components, the pineal and parapineal organs and the dorsal sac (DS). The pineal organ was composed of a short, thin pineal stalk (PS), vertically disposed with respect to the upper surface of the telencephalon, and a pineal vesicle (PV), located at the distal end of the PS and attached to the skull by connective tissue. The pineal window (PW), a site in the skull where the luminal information accesses the pineal organ, appeared just above the latter structures. In the epidermis of P. axelrodi's PW, club cells were identified, but were not observed in the epidermis of A. anisitsi's one. With respect to the DS, it appeared to be folded on itself, and was bigger and more folded in A. anisitsi than in P. axelrodi. Immunohistochemical assays revealed the presence of cone opsin-like and rod opsin-like photoreceptor cells in the PS and PV. These results provide a first insight into the morphological assembly of the pineal complex of both species, and contribute to a better understanding of the integration and transduction of light stimuli in characids. J. Morphol. 277:1355-1367, 2016. © 2016 Wiley Periodicals, Inc.

Adenosine triphosphate inhibits melatonin synthesis in the rat pineal gland.

Adenosine triphosphate (ATP) is released onto the pinealocyte, along with noradrenaline, from sympathetic neurons and triggers P2Y1 receptors that enhance ß-adrenergic-induced N-acetylserotonin (NAS) synthesis. Nevertheless, the biotransformation of NAS into melatonin, which occurs due to the subsequent methylation by acetylserotonin O-methyltransferase (ASMT; EC 2.1.1.4), has not yet been evaluated in the presence of purinergic stimulation. We therefore evaluated the effects of purinergic signaling on melatonin synthesis induced by ß-adrenergic stimulation. ATP increased NAS levels, but, surprisingly, inhibited melatonin synthesis in an inverse, concentration-dependent manner. Our results demonstrate that enhanced NAS levels, which depend on phospholipase C (PLC) activity (but not the induction of gene transcription), are a post-translational effect. By contrast, melatonin reduction is related to an ASMT inhibition of expression at both the gene transcription and protein levels. These results were independent of nuclear factor-kappa B (NF-kB) translocation. Neither the P2Y1 receptor activation nor the PLC-mediated pathway was involved in the decrease in melatonin, indicating that ATP regulates pineal metabolism through different mechanisms. Taken together, our data demonstrate that purinergic signaling differentially modulates NAS and melatonin synthesis and point to a regulatory role for ATP as a cotransmitter in the control of ASMT, the rate-limiting enzyme in melatonin synthesis. The endogenous production of melatonin regulates defense responses; therefore, understanding the mechanisms involving ASMT regulation might provide novel insights into the development and progression of neurological disorders since melatonin presents anti-inflammatory, neuroprotective, and neurogenic effects.

Morphologic changes in rat's pineal gland after eliminating retinal photic stimulation / Cambios morfológicos en la glándula pineal de ratas luego de la eliminación de la estimulación fótica retiniana

Melatonin secretion from mammalian pineal glands is regulated by light stimulation by means of a complex neuroanatomical pathway that includes the retina, hypothalamic suprachiasmatic nucleus, intermediolateral nucleus of the thoracic spinal cord, and finally, the superior cervical ganglia. The purpose of this study was to analyze the changes in the pinealocytes and the blood vessel density of the pineal gland after eliminating photic stimulation in rats. Thirteen adult Sprague-Dawley rats were divided into 2 groups, Group I acted as control, and Group II was subjected to a retinal lesion, by means of alcohol injected bilaterally to both ocular bulbs. After 3 weeks, the glands of both groups were processed with hematoxilin-eosin (HE) and observed with an optic microscope. Group II results presented higher values in the number of pinealocytes and in the blood vessels observed. The differences with Group I was significant at p <0.01. These results give an indirect evidence of the effect that photic stimulation suppression has in the pineal gland in rats.

La secreción de melatonina por parte de glándula pineal de los mamíferos es regulada por la estimulación luminosa mediante complejas vías neuro anatómicas que incluyen la retina, el núcleo supraquiasmático hipotalámico, el núcleo intermediolateral de la médula torácica y finalmente el ganglio cervical superior. El propósito de este estudio fue analizar los cambios en la densidad de pinealocitos y vasos sanguíneos de la glándula pineal, luego de eliminar la estimulación fótica en ratas. Se utilizaron 13 ratas adultas Sprague Dawley divididas en 2 grupos, Grupo I actuó como control, y el Grupo II fue sometido a una lesión retiniana, por medio de alcoholización bilateral de ambos bulbos oculares. Luego de tres semanas las glándulas de ambos grupos fueron procesadas para hematoxilina-eosina y observadas al microscopio óptico. El grupo II presentó valores mayores en el número de pinealocitos y de vasos sanguíneos observados, las diferencias con el Grupo I fueron significativas con p<0.01. Estos resultados entregan una evidencia indirecta del efecto que la supresión de la estimulación fótica tiene en la glándula pineal en ratas.