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.     

Mitotic activity and its 24-hour rhythm in the rat pineal gland

Previous studies have yielded equivocal results concerning the 24-hour rhythmicity of mitotic activity in the rat pineal. The aim of the present study was to re-investigate this problem by carrying out three separate 24-hour experiments on alternate days. The results obtained confirm previous findings showing that in the pineal gland of adults mitotic activity is low. On average 22.3 mitotic figures of pinealocytes are seen per pineal gland, corresponding to a mitotic index of 0.2-0.6/1,000 pinealocytes. Mitotic activity is distinctly higher at daytime than at night. The timing of the peaks and troughs differs slightly from experiment to experiment. The majority of observations now indicate that in the rat pineal gland mitotic activity is higher at day time than at night.     

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.     

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.     

Estradiol modulation of pineal gland activity in the wild bandicoot rat, Bandicota bengalensis

In the present study, the effect of estradiol dipropionate on the cytology and mitotic activity of the pineal gland was evaluated in adult ovariectomized and juvenile bandicoot rats, Bandicota bengalensis. Estradiol treatment for 5 days inhibited the ovariectomy-induced hypertrophy of the pineal gland, and increased the nuclear diameters of pinealocytes in juvenile males while having no effect on the pineal cytology of juvenile females. Estradiol injection induced mitosis in the pinealocytes of adult ovariectomized and juvenile bandicoot rats. Thus, estradiol exhibited a dual action (stimulatory and inhibitory) on the pineal gland of this wild rat.     

Microglia play a role in mediating the effects of cytokines on the structure and function of the rat pineal gland

The role of the pineal gland in regulating immune function has been extensively investigated. However, there is little information about possible feedback mechanisms of immunological factors on pineal gland neuroendocrine functions. Therefore, experiments were designed to test the effects of cytokines (interferon-gamma, IFN-gamma, interleukin-1 beta, IL-1 beta; tumor necrosis factor-alpha, TNF-alpha; transforming growth factor-beta 1, TGF-beta 1) on pinealocytes and the role of pineal microglia in mediating these cytokine effects in the pineal gland of the rat. Our studies showed that IFN-gamma enhanced 5-hydroxytryptamine (5-HT) content (measured by high-performance liquid chromatography, HPLC) and increased pinealocyte process length in pineal cultures. IL-1 beta treatment decreased 5-HT content in both cell and organ culture, but exhibited no effect on pinealocyte process length. 5-HT content and process length were decreased by TNF-alpha treatment. IFN-gamma and IL-1 beta exhibited no significant effect in the absence of microglia in cell cultures. In contrast, TNF-alpha caused a further decline in 5-HT content even in the absence of microglia in the cultures. The effects of TNF-alpha were probably due to toxic effects, since an increased number of pyknotic nuclei were observed in treated cultured explants. TGF-beta 1 treatment caused aggregation of pinealocytes in cultures and suppressed process length and 5-HT content. In conclusion, cytokine effects on pinealocytes may be mediated by microglia (IFN-gamma and IL-1 beta) or act directly on pinealocytes (TNF-alpha). The presence of IL-1 beta and TGF-beta 1 protein in the pineal gland and the suppressive effect of TGF-beta 1 on pinealocytes in cultures further suggest that endogenous cytokines play regulatory roles in response to peripheral homeostatic changes.     

Cholinergic signaling in the rat pineal gland

Summary 1. Innervation of the mammalian pineal gland is mainly sympathetic. Pineal synthesis of melatonin and its levels in the circulation are thought to be under strict adrenergic control of serotoninN-acetyltransferase (NAT). In addition, several putative pineal neurotransmitters modulate melatonin synthesis and secretion.2. In this review, we summarize what is currently known on the pineal cholinergic system. Cholinergic signaling in the rat pineal gland is suggested based on the localization of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE), as well as muscarinic and nicotinic ACh binding sites in the gland.3. A functional role of ACh may be regulation of pineal synaptic ribbon numbers and modulation of melatonin secretion, events possibly mediated by phosphoinositide (PI) hydrolysis and activation of protein kinase C via muscarinic ACh receptors (mAChRs).4. We also present previously unpublished data obtained using primary cultures of rat pinealocytes in an attempt to get more direct information on the effects of cholinergic stimulus on pinealocyte melatonin secretion. These studies revealed that the cholinergic effects on melatonin release are restricted mainly to intact pineal glands since they were not readily detected in primary pinealocyte cultures.     

Cytochemical localization of type-A and -B monoamine oxidase in the rat pineal gland

Summary In the mammalian pineal gland, serotonin (5-HT) is located both in the pinealocytes and in the noradrenergic nerve terminals. Pineal 5-HT can be metabolized by three different routes, one of these being its deamination, catalized by monoamine oxidase (MAO). MAO is known to exist as two isozymes, MAO-A and MAO-B. Using two different cytochemical methods at the ultrastructural level, we have localized the presence of MAO in the pineal gland of the rat. The use of selective inhibitors of A-type (clorgyline) and B-type (deprenyl) has shown that MAO-A is localized in the noradrenergic nerve terminals, while pinealocytes contain MAO-B. Taking into account that 5-HT is only deaminated by MAO-A, the specific association of each MAO isozyme with a defined cell type implicates that two cellular compartments are needed in the pineal gland for the biosynthesis of 5-methoxytryptophol and 5-methoxyindole acetic acid, while for the synthesis of melatonin and 5-methoxytryptamine just one cellular compartment, the pinealocyte, is appropriate.     

Expression of synaptic vesicle trafficking proteins in the developing rat pineal gland

Adult mammalian pinealocytes contain several synaptic membrane proteins that are probably involved in the regulation of targeting and exocytosis of synaptic-like microvesicles (SLMVs). Immunohistochemical techniques have now demonstrated the spatiotemporal expression pattern of some of these proteins during rat pineal ontogenesis. Various synaptic vesicle trafficking proteins are detectable in proliferating epithelial cells of the pineal anlage even at embryonic day 17.5 (E 17.5), with the exception of syntaxin I (weakly expressed from E 19.5) and dynamin I (whose levels increase markedly during the first postnatal week). Numerous cells exhibiting strong immunoreactivity for synaptobrevin II, SNAP-25, synaptophysin, and munc-18-1 are distributed throughout the increasingly compact gland at E 19.5 and E 20.5; however, their number declines toward the proximal deep part of the organ. Groups of postmitotic cells situated at the surface of the developing gland exhibit marked immunoreactivity for the aforementioned proteins and lie close to the laminin-immunoreactive outer limiting basement membrane or to its remnants in regions of basement membrane dissolution. We also show that synthesis of vimentin and S-antigen seems to begin earlier during pineal development than previously recognized. Thus, synaptic vesicle trafficking proteins are the earliest molecular markers of pinealocyte differentiation known to date, being expressed well before the onset of rhythmic hormone secretion in the pineal gland, where they may play a role in morphogenetic events. Components of the extracellular matrix such as laminin may be critically involved in the upregulation of synaptic membrane protein expression. The dynamin immunostaining pattern indicates that SLMVs of pinealocytes begin to undergo regulated cycles of exo/endocytosis during postnatal week 1.     

Expression of neuron-specific enolase in the pineal organ of the domestic fowl during post-hatching development

Immunohistochemistry for neuron-specific enolase (NSE) revealed that NSE is localized in both a limited number of pinealocytes and intrinsic afferent neurons in the pineal organ of the domestic fowl. Furthermore, a computer-assisted three-dimensional imaging technique allowed to clarify the reverse distributional pattern of both elements: NSE-positive pinealocytes displayed a dense distribution especially in the vesicular portion of the gland, whereas NSE-immunoreactive nerve cells were mainly found in the pineal stalk. The number of NSE-positive intrinsic neurons in the pineal organ of chickens decreased rapidly after hatching, with a concentration of these elements in the basal portion (stalk) of the pineal organ. On the other hand, immunoreactive pinealocytes increased remarkably in the end-vesicle of the organ with age, followed by a gradual expansion toward the proximal portion. Thus, the spectacular increase in NSE-positive pinealocytes and the progressive reduction of reactive neurons occurred in parallel during the course of post-hatching development. NSE-immunoreactive pinealocytes displayed morphological characteristics of bipolar elements, endowed with an apical protrusion into the pineal lumen and a short basal process at younger stages, whereas multipolar types of NSE-positive pinealocytes were predominantly found in the adult domestic fowl. These results indicate that in the pineal organ of the domestic fowl (1) the ontogenetic expansion of NSE-immunoreactive pinealocytes is paralleled by a regressive afferent innervation, (2) the NSE-positive pinealocytes transform from a bipolar (columnar) type to a multipolar type during post-hatching development, and (3) these ontogenetic changes in the NSE-immunoreactivity and morphology of pinealocytes may reflect the development of a neurosecretory-like capacity of the organ.     

Neuronal markers in the rodent pineal gland--an immunohistochemical investigation

Although some embryological and morphological features speak in favour of a neuronal character of rodent pinealocytes, histochemistry and ultrastructure let this issue appear controversial. Using antibodies to different neurofilaments, the neural adhesion molecule L1, synaptophysin and tubulin as neuronal markers, the pineal glands of rat and guinea-pig were studied by means of immunofluorescence. Neurofilament-immunoreactivity was present in some rat pineal nerve fibers and in the majority of guinea-pig pinealocytes, L1 decorated rat intrapineal nerve fibers, synaptophysin was almost ubiquitously distributed in the pineal of both species, while tubulin-immunofluorescence was seen in nerve fibers of rat and guinea-pig pineal and in some pinealocytes of the latter. These findings speak in favour of the neuronal character of guinea-pig pinealocytes. The lack of neurofilament- and tubulin-immunoreactivity in rat pinealocytes might be attributable to very low concentrations of these proteins or species differences as to their expression. Further studies including in situ-hybridisation of relevant mRNAs will be necessary to answer these questions definitely.     

Nucleolus-like bodies in the pineal gland of the Djungarian hamster (Phodopus sungorus)

Summary Light- and electron-microscopic observations on the pineal gland of Phodopus sungorus revealed intracytoplasmic inclusions resembling nucleolus-like bodies similar to those found in other regions of the central nervous system. Bernhard's EDTA method was used to confirm that these inclusions were nucleolus-like bodies. These structures were rarely found in pinealocytes of sexually active longday animals, whereas large numbers of them were observed in pinealocytes of sexually quiescent short-day animals. Nucleolus-like bodies may therefore be involved in pineal secretion.     

Ultrastructure of the rat pineal stalk

The ultrastructure of the rat pineal stalk was described. The pineal stalk contained few pinealocytes, glial cells and numerous nerve fibers. The last were mostly non-myelinated axons, although a few myelinated ones were also observed. Glial cells showed many filaments, mostly in the processes which presented a longitudinal orientation. Other more lamellar processes were found enclosing the axons. The pineal stalk became wider as it reached the body of the gland. Ultrastructurally, this wide region resembled more the pineal body. Bundles of non-myelinated nerve fibers were seen around the pineal stalk.     

Ultrastructural changes in the rat pineal gland after sympathetic denervation. Quantitative study

Ultrastructural changes in the rat pineal gland were studied quantitatively 7 and 60 days after the sympathetic denervation by bilateral excission or decentralization of superior cervical ganglia. The surface occupied by pineal parenchymal cells decreased in rats of experimental groups with respect to the control group. Furthermore, profile areas of the cytoplasm, nucleus and nucleolus of the pinealocytes were also diminished. Cytoplasmic lipid droplets in the pinealocytes were markedly decreased in number and size in experimental rats. As demonstrated by the Kruskal-Wallis H test, statistically significant differences were found between rats of the control and operated groups. Rats treated by superior cervical ganglionectomy or decentralization showed morphological changes indicating a hypofunctional pineal gland, although differences were found between both groups.     

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.     

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 epiphysis (pineal body) and the APUD system

Light and electron microscopic studies were conducted on 10 humans who died of the different cardiac diseases; and 20 guinea pigs pineal glands. Pinealocytes or secretory cells of the pineal gland have morphological likeness with the APUD system cells. They have a well-developed endoplasmic reticulum, Golgi complex, mitochondrial component and in cytoplasm dense-core vesicles are discovered. However the pinealocytes have a neuron-like structure and they are not separate cells as apudocytes, but they are a principal component of the pineal parenchyma in which pinealocytes are in tight interactions with glia, blood vessels and nerve terminations. Analysis of morphological and functional similarity and difference between pinealocytes and apudocytes allows to consider pineal gland as an APUD organ. A circadian rhythmicity of some secretory vesicles in pinealocytes of the guinea pig has been established.     

Cells expressing preproenkephalin mRNA in the rat pineal gland are not serotonin-producing pinealocytes: Evidence usingin Situ hybridization combined with immunocytochemistry for serotonin

Summary 1. Preproenkephalin (PPEnk) mRNA expressing cells have been identified in rat pineal gland using radioactivein situ hybridization histochemistry. 2. Approximately 7% of the cells in the pineal gland (7.5±0.86, mean ± 95% CI) express PPEnk mRNA. These cells are distributed throughout the pineal as either scattered single cells or small groups of cells with large round or oval nuclei. 3. Usingin situ hybridization combined with ABC immunocytochemistry for serotonin (5-HT) in the same pineal sections, the PPEnk mRNA labeling cells are found not to be serotonin-immunoreactive cells. These data indicate that the PPEnk mRNA is expressed in a certain discrete subpopulation of cells in the rat pineal gland and these cells are not serotonin-producing pinealocytes. 4. The physiologic role of PPEnk-derived peptides in the pineal remains unknown. It is possible that these peptides either are synthesized and secreted as hormones or act as pineal paracrine signals.     

Effect of photoperiod on pineal gland volume and pinealocyte size in the Chinese hamster, Cricetulus griseus

Male adult (200-day-old) Chinese hamsters (Cricetulus griseus) raised from weaning under either LD 16:8 or LD 8:16 were used. The pineal gland of the Chinese hamster consists of superficial (major) and deep (minor) components and a continuous, or interrupted, narrow parenchymal stalk interposed between them. The volume of the superficial pineal including the parenchymal stalk is greater under LD 16:8 than under LD 8:16. Under both photoperiods, pinealocytes in the superficial pineal have larger nuclei and more abundant cytoplasm than those in the deep pineal. Nuclei in the superficial pineal appear pale and usually have irregular profiles, whereas those in the deep pineal appear dark and have round profiles. In the superficial pineal, pinealocyte nuclei are larger, paler, and more irregular; and, in addition, nuclear density is lower under LD 16:8 than under LD 8:16. Similar, but less prominent, photoperiod-induced changes occur in the volume of the deep pineal, the size of pinealocytes, and pinealocyte nuclear morphology in the deep pineal. The results indicate that the development and differentiation of pinealocytes in both pineal portions may be advanced under long photoperiods and delayed under short photoperiods, although pinealocytes in the deep pineal may remain not fully differentiated even in adults. Since testicular weights and body weights are similar under both photoperiods, the photoperiod may exert marked influences on the development of the pineal gland without affecting reproductive activity and growth rates of animals.     

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.