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.

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.

Microstructure and morphoquantitative study of pineal gland in Santa Ines sheep / Estudo morfoquantitativo e da microestrutura da glândula pineal em ovinos Santa Inês

This study examined morphoquantitative aspects of the pineal gland in Santa Ines sheep in anoestrus physiological and reproductive activity. Seven female adult sheep collected from slaughterhouses in the Brasilia-DF region, with no clinical signs related to diseases of the nervous system were used. The brains were removed and their dimensions were measured. The brains were sectioned to expose and measure the pineal gland. The fragments of the diencephalon containing the pineal gland were immersed in 20% formaldehyde solution. Subsequently, the fragments were submitted to conventional histological techniques. The Hematoxylin-eosin, Toluidine blue and Gomori trichrome staining were used for a morphoquantitative analysis. The brain had an average length of 67.25 ± 1.75 mm and average width of 58.97 ± 4.0 mm. The pineal gland had an average length of 6.98 ± 0.79 mm and average width of 6.40± 1.35 mm. The quantitative analysis showed an average 86.27 ± 30.44 of pinealocytes per field. According to the Pearson correlation test, the number of pinealocytes showed a weak negative linear correlation (r = -0.11) with the length of the pineal gland and a weak positive linear correlation (r = 0.39) with the width. Therefore, the number of pinealocytes has a stronger correlation with the width of the gland compared to the length. Mastocytes were present in only one animal (14.28%). Calcareous concretions were observed in two animals (28.57%). The connective tissue formed the capsule surrounding the gland with no projections into the parenchyma.

O presente estudo avaliou os aspectos morfoquantitativos e qualitativos da glândula pineal em fêmeas de ovinos da raça Santa Inês em atividade reprodutiva e anestro fisiológico. Foram utilizados sete ovinos, fêmeas e adultas, coletados em frigoríficos da região de Brasília-DF. Estes não apresentavam sinais clínicos relacionados a afecções do sistema nervoso. Os encéfalos foram retirados e dimensões mensuradas. Posteriormente foram seccionados para expor a glândula pineal e mensurar suas dimensões. Os fragmentos do diencéfalo, contendo a glândula pineal, foram submersos em solução aquosa a 20% de formaldeído e posteriormente submetidos a técnicas histológicas convencionais. Para a avaliação morfoquantitativa desta glândula, foram empregadas as colorações Hematoxilia-Eosina, Azul de Toluidina e Tricrômio de Golmori. O encéfalo teve um comprimento médio de 67,25 ± 1,75 mm e a largura média de 58,97 ± 4,0 mm. Já a glândula pineal apresentou o comprimento médio de 6,98 ± 0,79 mm e a largura média de 6,40 ± 1,35 mm. Diante da análise microscópica, foi obtida uma média de 86,27 ± 30,44 pinealócitos por campo. Ao ser aplicado o teste de correlação de Pearson, o número de pinealócitos apresentou fraca correlação linear negativa (r = -0,11) em relação ao comprimento da glândula pineal e uma fraca correlação linear positiva (r = 0,39) em relação à largura da mesma. Portanto o número de pinealócitos possui uma maior relação com a largura da glândula do que com o comprimento. Mastocitos estiveram presentes em apenas um animal (14,28%) e as concreções calcareas foram observadas em dois animais (28,57%). A glândula se mostrou envolta por uma cápsula de tecido conjuntivo com ausência de projeções para o parênquima.

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.

Morphologic study of the pineal gland of the dog / Estudo morfológico da glândula pineal do cão

Following the discovery of the melatonin by Lerner et al. (1958), new research and perspectives were developed in order to improve the knowledge regarding the pineal gland. This hormone is not only related to the circadian control but also influences other behavioral functions such as the reproductive cycle and thermoregulation. In this study the morphology of the pineal gland (epiphysis cerebri) from 20 dogs were analyzed by means of macroscopic and microscopic evaluation. The shape of the gland ranged from conic to tongue-like shape (in relation to human tongue). The gland color ranged from beige to gray-brownish and it had a gelatinous consistency. The width and length of the glands ranged from 1.38 to 2.39 mm and 1.53 to 2.96 mm, respectively. Capsule, septa, pinealocytes, glial cells and pigment granules were microscopically found in all glands. No calcareous concretions were observed.

Após a descoberta da melatonina por Lerner et al. (1958), novas pesquisas e perspectivas surgiram tornando a pineal uma estrutura mais estudada. Além disso, sabe-se que este hormônio está relacionado com o controle circadiano e outras funções comportamentais fisiológicas como o ciclo reprodutivo e termorregulação. Neste trabalho, foram utilizados 20 cães e estudamos a morfologia das glândulas, empregando uma análise macroscópica e microscópica. O formato das pineais variou de cônico a glossóide (em relação à língua humana); cor entre bege e marrom-acinzentado e apresentaram consistência gelatinosa. A largura e o comprimento das glândulas variaram respectivamente de 1,38 a 2,39 mm e de 1,53 a 2,96 mm. Quanto à microscopia, utilizamos coloração de eosina-hematoxilina (HE), e em todas as pineais observamos a presença dos seguintes componentes: cápsula; septos ou trabéculas pinealócitos; células da glia; grânulos de pigmentos, e não foram notadas concreções calcáreas.

Morphologic study of the pineal gland of the dog / Estudo morfológico da glândula pineal do cão

Following the discovery of the melatonin by Lerner et al. (1958), new research and perspectives were developed in order to improve the knowledge regarding the pineal gland. This hormone is not only related to the circadian control but also influences other behavioral functions such as the reproductive cycle and thermoregulation. In this study the morphology of the pineal gland (epiphysis cerebri) from 20 dogs were analyzed by means of macroscopic and microscopic evaluation. The shape of the gland ranged from conic to tongue-like shape (in relation to human tongue). The gland color ranged from beige to gray-brownish and it had a gelatinous consistency. The width and length of the glands ranged from 1.38 to 2.39 mm and 1.53 to 2.96 mm, respectively. Capsule, septa, pinealocytes, glial cells and pigment granules were microscopically found in all glands. No calcareous concretions were observed.

Após a descoberta da melatonina por Lerner et al. (1958), novas pesquisas e perspectivas surgiram tornando a pineal uma estrutura mais estudada. Além disso, sabe-se que este hormônio está relacionado com o controle circadiano e outras funções comportamentais fisiológicas como o ciclo reprodutivo e termorregulação. Neste trabalho, foram utilizados 20 cães e estudamos a morfologia das glândulas, empregando uma análise macroscópica e microscópica. O formato das pineais variou de cônico a glossóide (em relação à língua humana); cor entre bege e marrom-acinzentado e apresentaram consistência gelatinosa. A largura e o comprimento das glândulas variaram respectivamente de 1,38 a 2,39 mm e de 1,53 a 2,96 mm. Quanto à microscopia, utilizamos coloração de eosina-hematoxilina (HE), e em todas as pineais observamos a presença dos seguintes componentes: cápsula; septos ou trabéculas pinealócitos; células da glia; grânulos de pigmentos, e não foram notadas concreções calcáreas.