Cell proliferation in the developing rat pineal gland. A bromodeoxyuridine immunohistochemical study

The immunohistochemical detection of bromodeoxyuridine (BrdU) was used to study the cell proliferation in the developing rat pineal gland, from the appearance of pineal primordium in the embryonic day 15 (E15) until 30 days after birth. The results showed three different proliferative phases. From E15 to E21, the pineal gland shows a phase of rapid proliferation. The second phase corresponds to the first postnatal week, in which the number of labeled cells per surface unit decreases suddenly to values between 20% to 10% of those of embryonic period. From the second postnatal week onwards, the number of BrdU-positive cells progressively decreases.     

Postnatal development of the rabbit pineal gland. A light- and electron-microscopic study.

The development of the rabbit pineal gland has been studied by light and electron microscopy from the 1st to the 120th postnatal day. After 24 h of postnatal life, the pineal parenchyma is highly cellular, showing two identifiable cell types: pinealocytes I and II. Immature type II pinealocytes arrange either in cellular cords or clusters or forme rosette-like structures. At the 5th postnatal day, corticomedullar differentiation is established. Rosette-like structures and cellular cords are absent from the cortex. Along the postnatal period, nuclei of pinealocytes are set apart due to cytoplasmic widening and development of cell processes. These structures pervade the cellular cords and rosette-like structures formed by immature type II pinealocytes. Rosette-like structures are no longer seen beyond the 30th postnatal day, and cords of type II pinealocytes from the 90th postnatal day on. At this time, the rabbit pineal gland is considered to be histologically mature.     

Glia-pinealocyte network: the paracrine modulation of melatonin synthesis by tumor necrosis factor (TNF)

The pineal gland, a circumventricular organ, plays an integrative role in defense responses. The injury-induced suppression of the pineal gland hormone, melatonin, which is triggered by darkness, allows the mounting of innate immune responses. We have previously shown that cultured pineal glands, which express toll-like receptor 4 (TLR4) and tumor necrosis factor receptor 1 (TNFR1), produce TNF when challenged with lipopolysaccharide (LPS). Here our aim was to evaluate which cells present in the pineal gland, astrocytes, microglia or pinealocytes produced TNF, in order to understand the interaction between pineal activity, melatonin production and immune function. Cultured pineal glands or pinealocytes were stimulated with LPS. TNF content was measured using an enzyme-linked immunosorbent assay. TLR4 and TNFR1 expression were analyzed by confocal microscopy. Microglial morphology was analyzed by immunohistochemistry. In the present study, we show that although the main cell types of the pineal gland (pinealocytes, astrocytes and microglia) express TLR4, the production of TNF induced by LPS is mediated by microglia. This effect is due to activation of the nuclear factor kappa B (NF-kB) pathway. In addition, we observed that LPS activates microglia and modulates the expression of TNFR1 in pinealocytes. As TNF has been shown to amplify and prolong inflammatory responses, its production by pineal microglia suggests a glia-pinealocyte network that regulates melatonin output. The current study demonstrates the molecular and cellular basis for understanding how melatonin synthesis is regulated during an innate immune response, thus our results reinforce the role of the pineal gland as sensor of immune status.     

Induction of c-fos protein-like immunoreactivity in the rat and hamster pineal gland after the onset of darkness

Induction of c-fos protein (FOS) after the onset of darkness was studied immunocytochemically in the rat and hamster pineal gland. The animals were kept on a 12:12 h light-dark cycle. Before the dark period no FOS staining was seen in either rat or hamster pineal cells. Five hours after the onset of darkness 342 +/- 18 pinealocytes/0.2 mm2 (mean +/- SD) displayed FOS-like immunoreactivity in the hamster pineal gland; in the rat pineal gland only 5 +/- 2 pinealocytes/0.2 mm2 showed a faint staining. Two hours later the density of FOS positive cells was decreased to 60 +/- 11/0.2 mm2 in the hamster but increased to 519 +/- 103/0.2 mm2 in the rat pineal gland. Three hours before the beginning of the light period no FOS positive cells were detected in either animal. Both the rat and hamster pineal gland showed a transient and temporally defined expression of c-fos protein in the middle of the dark period. This may be related to a more active functional state of pinealocytes, which is reflected in a peak of melatonin synthesis during the darkness.     

Induction of c-fos protein-like immunoreactivity in the rat and hamster pineal gland after the onset of darkness

Summary Induction of c-fos protein (FOS) after the onset of darkness was studied immunocytochemically in the rat and hamster pineal gland. The animals were kept on a 12:12 h light-dark cycle. Before the dark period no FOS staining was seen in either rat or hamster pineal cells. Five hours after the onset of darkness 342±18 pinealocytes/0.2 mm² (mean±SD) displayed FOS-like immunoreactivity in the hamster pineal gland; in the rat pineal gland only 5±2 pinealocytes/0.2 mm² showed a faint staining. Two hours later the density of FOS positive cells was decreased to 60±11/0.2 mm² in the hamster but increased to 519±103/0.2 mm² in the rat pineal gland. Three hours before the beginning of the light period no FOS positive cells were detected in either animal. Both the rat and hamster pineal gland showed a transient and temporally defined expression of c-fos protein in the middle of the dark period. This may be related to a more active functional state of pinealocytes, which is reflected in a peak of melatonin synthesis during the darkness.     

Structure and Function of the Vertebrate Pineal Gland

This review presents data from the literature on structure and function of the pineal gland. Discussed are the histological and ultrastructural characteristics of the gland, its function according to novel results, peculiarity of synthesis and secretion of melatonin and its function, as well as the role of the pineal gland in circadian organization of organisms. The problems of evolution of the pineal function in the row of vertebrates are considered.     

Annual variations of the NPYergic innervation of the pineal gland in the European hamster (Cricetus cricetus): a quantitative immunohistochemical study

A prominent innervation of the pineal gland of the European hamster with nerve fibres containing neuropeptide Y (NPY) and tyrosine hydroxylase (TH) was demonstrated by means of immunohistochemistry. Nearly all the TH- and NPY-immunoreactive nerve fibres in the superficial pineal gland disappeared after bilateral superior cervical ganglionectomy, showing that the majority of NPY- and TH-immunoreactive nerve fibres belonged to the sympathetic nervous system. Since, in the European hamster, preliminary studies of the NPY-fibre density in the pineal gland had indicated seasonal changes, the density of NPY-immunoreactive nerve fibre profiles was ascertained in the superficial pineal gland in a series of animals between the first part of November and late April. The highest density of NPY-immunoreactive nerve fibre profiles was observed during midwinter. On the other hand, during the same period of the year, the number of sympathetic TH-immunoreactive sympathetic nerve fibre profiles did not exhibit seasonal variation, nor did substitution of testosterone, during the sexually inactive period, affect the density of NPY-containing nerve fibres in the gland. Our results show the presence of a testosterone-independent annual variation in the content of NPY in the sympathetic nerve fibres innervating the pineal gland of the European hamster. This variation can be correlated with the changes in the daily pattern of melatonin production observed by others in the same species at this period of the year.     

Age-related morphometric changes in the pineal gland. A comparative study between C57BL/6J and CBA mice

Relatively little is known about the effects of melatonin on the aging of the pineal, the organ which is the main place for synthesis of this hormone. Using simple morphometric methods, some parameters of the pineal gland, such as total volume, number of pinealocytes and pinealocyte volume were estimated in two mice strains: normal CBA and melatonin-deficient C57BL/6J. Two age groups, 6 weeks and 10 months, were studied in order to evaluate possible differential age-related changes between both strains. Pineals of both strains have similar morphometric and morphological features at 6 weeks of age. This suggests that pineal development, which has already concluded at 6 weeks of age, is not affected by the absence of melatonin synthesis in the pinealocytes. Later on, CBA pineal showed an increase in size caused by cellular hypertrophy. In contrast, the C57BL/6J pineal volume decreased by loss of pinealocytes in the same period of time. Semithin sections analysed by light microscopy did not show that this cell death was evident in the C57BL/6J strain at any of the ages studied. Thus, a gradual loss of pinealocytes could be hypothesised in these pineals. These results suggest that pineal melatonin could have a role in the maintenance of pinealocyte viability and the increase of pineal size which takes place after development. The abnormal pattern observed in the C57BL/6J pineal should be taken into account in future studies on this gland.     

Regional distribution and cellular expression of tryptophan hydroxylase messenger RNA in postmortem human brainstem and pineal gland

The characterization and cellular localization of tryptophan hydroxylase mRNA in the human brainstem and pineal gland were investigated by using northern blot analysis and in situ hybridization histochemistry. Northern analysis of human pineal gland revealed the presence of two mRNA species that were absent in RNA isolated from human raphe. In situ hybridization experiments revealed very dense hybridization signal corresponding to tryptophan hydroxylase mRNA in cells throughout the pineal gland. In contrast, tryptophan hydroxylase mRNA was heterogeneously distributed in neurons in the dorsal and median raphe nuclei. Within the dorsal raphe, the ventrolateral and interfascicular subnuclei contained the greatest number of tryptophan hydroxylase mRNA-positive neurons. Also, the cellular concentration of tryptophan hydroxylase mRNA varied widely within the dorsal and median raphe. Comparison of the cellular concentration of tryptophan hydroxylase mRNA between the pineal gland and the raphe nuclei revealed an 11- and 46-fold greater average grain density of tryptophan hydroxylase mRNA positive cells in the pineal gland compared with the dorsal and median raphe, respectively. These findings are the first to demonstrate the cellular localization of tryptophan hydroxylase mRNA in the human brain and pineal gland as well as heterogeneity in the cellular concentration within and between these tissues.     

The effect of the transplanted pineal gland on the sympathetic innervation of the rat sublingual gland

We investigated the effect of the pineal on sympathetic neurons that normally innervate the sublingual gland of the rat. When the pineal gland was transplanted into the sublingual gland, it remained as a distinct mass that was innervated by sympathetic axons. Injection of the retrograde tracer, Fast Blue, into the sublingual gland labelled sympathetic neurons in the ipsilateral superior cervical ganglion (SCG). Thirty per cent of all neurons labelled retrogradely by Fast Blue injection into transplanted pineal glands were immunoreactive for both neuropeptide Y (NPY) and calbindin. This combination is characteristic of sympathetic neurons innervating the pineal gland in its normal location, but not the sympathetic vasoconstrictor neurons normally innervating the sublingual gland. This, and our previous study in which the pineal gland was shown to similarly influence the phenotype of salivary secretomotor neurons, suggests that a range of different functional classes of sympathetic neuron are able to change their phenotype in response to signals released by the pineal gland.This work was supported by Project Grant No. 145634 from the National Health and Medical Research Council of Australia     

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.     

The pineal gland and seasonal reproductive adjustments

The pineal gland seems to play a major role in controlling and synchronizing circannual reproductive cycles in some mammals. The following scheme is based primarily on experimental evidence compiled using the golden hamster. However, it is probably applicable in one form or another to a number of long day breeding species. When hamsters are kept under natural photoperiodic conditions they exhibit a period of infertility followed by a period of fertility. The entire cycle encompasses approximately one year. The cycle has been divided into 4 distinctive phases: the inhibition phase, the sexually quiescent phase, the restoration phase, and the sexually active phase. During the inhibition phase the decreasing photoperiods in the fall of the year cause activation of the pineal gland and, as a consequence, gonadal regression. The sexually quiescent phase requires an intact pineal gland to maintain the gonads in a non-functional state. The restoration phase, which occurs in the spring of the year, allows the gonads to become recrudescent. This phase of the cycle seems to be light independent. The sexually active phase extends from spring until fall. During this phase the hypothalamo-pituitary-gonadal axis seems to be refractory to inhibition by the pineal gland. Some light is required during the summer months to interrupt the refractory period.     

Development and Regulation of Rhodopsin Kinase in Rat Pineal and Retina

Rhodopsin kinase, once thought to be a retinal enzyme, was recently found at high levels in the pineal gland. In the present study the developmental pattern and the regulation by environmental lighting of this enzyme in both tissues was studied in the rat. Enzyme activity was present in the neonatal pineal gland several days earlier than in the retina, and increased gradually up to 20 days of age and remained at that level thereafter; the retinal enzyme appeared to increase until day 60. Pineal and retinal rhodopsin kinase activities showed a 25% increase in in the middle of the dark and the beginning of the light period, respectively. Exposure to constant light caused a 50% decrease in rhodopsin kinase levels in both tissues. However, only pineal rhodopsin kinase activity declined followed bilateral superior cervical ganglionectomy. This indicates pineal rhodopsin kinase activity is similar to other pineal enzymes in that it is controlled by light acting through the sympathetic nervous system. In contrast, the light-induced decrease in retinal rhodopsin kinase may be due to the direct destructive effect of light on the retina. The finding of neural control of pineal rhodopsin kinase in the pineal gland of adult rats is consistent with a function of the enzyme in the neural regulation of pineal function.     

Embryonic development of the bovine pineal gland (Bos taurus) during prenatal life (30 to 135 days of gestation)

The ontogenesis of the pineal gland of 30 bovine embryos (Bos taurus) has been analysed from 30 until 135 days of gestation by means of optical microscopy and immunohistochemical techniques. For this study, the specimens were grouped into three stages in accordance with the most relevant histological characteristics: Stage 1 (30 to 64 days of prenatal development); Stage 2 (70 to 90 days) and Stage 3 (106 to 135 days). In the cow, it is from 30 days of gestation that the first glandular outline becomes differentiated from the diencephalic ependyma of the third ventricle. This differentiation includes the phenomena of proliferation and multiplication of the ependymal cells that form the epithelium of the pineal outline in development. At 82 days of intrauterine life, in the interior of the pineal parenchyma, we witnessed some incipient pseudoglandular structures that at 135 days were well differentiated. The pineal parenchyma displays a cytology made up of two cellular types of structurally distinct characteristics: pinealoblasts and interstitial cells. Both cellular types begin differentiation at 70 days of embryonic development, the pinealoblasts being greater in number than the interstitial cells. The glandular stroma is formed from the capsular, trabecular and the perivascular connective tissue, filling the interparenchymal space. A dense network of capillaries, which drive across the trabecular connective tissue towards the central glandular zone where their density increases and their calibre is reduced, complete the glandular structure. GFAP positive cells were observed in the embryonic pineal parenchyma in stage 3. At 135 days of gestation, NPY positive fibers entered the pineal gland through the pineal capsule occupying a perivascular localization. Morphological studies of this nature are vital for future use as parameters, indicative of the functional activity of the bovine pineal gland during embryonic development.     

Indications for the presence of two populations of serotonin-containing pinealocytes in the pineal complex of the golden hamster (Mesocricetus auratus)

Summary Serotonin-like immunoreactivity was investigated in the pineal complex of the golden hamster by use of the indirect immunohistochemical technique. The superficial and deep portions of the pineal gland, and also the pineal stalk exhibited an intense cellular immunoreaction for serotonin. In addition, perivascular serotonin-immunoreactive nerve fibers were observed. Some serotonin-immunoreactive processes of the pinealocytes terminated on the surface of the ventricular lumen in the pineal and suprapineal recesses, indicating a receptive or secretory function of these cells. Several serotonin-immunoreactive processes connected the deep pineal with the habenular area. One week after bilateral removal of both superior cervical ganglia the serotonin immunoreaction of the entire pineal complex was greatly decreased. However, some cells in the pineal complex, of which several exhibited a neuron-like morphology, remained intensively stained after ganglionectomy. This indicates that the indoleamine content of some cells in the pineal complex of the golden hamster is independent of the sympathetic innervation.Supported by a Grant from the Italian Society for Veterinary Sciences     

Diurnal variations of S-adenosyl-L-methionine and adenosine content in the rat pineal gland

S-adenosylmethionine and adenosine levels in the rat pineal gland were determined by high-performance liquid chromatography after fractionation of the pineal extracts. The concentration of S-adenosylmethionine follows a circadian rhythm and is about three times higher during the day (2.5 nmol/gland) than the night (1.1 nmol/gland). The variations in the level of adenosine are apparently more complex. Over the 24 hours period there are two maxima at 03.00 (120 pmol/gland) and 15.00 hrs (100 pmol/gland) and one minimum at 09.00 hrs (50 pmol/gland). In addition, only an ultradian rhythm with a period of 12 hrs and an acrophase of 3 hrs can be evinced by computer analysis.     

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.