The presence of acetylcholinesterase-positive nerve fibres in the deep pineal gland and the pineal stalk but not in the superficial part of adult albino rats

Application of the histochemical method for testing acetylcholinesterase (AChE, EC 3.1.1.7) showed the presence of AChE-positive nerve fibers in the deep pineal gland and the pineal stalk but not in the superficial part of adult albino rats. These findings may indirectly support the existence of the potentially cholinergic innervation of at least some of the rat pinealocytes present in these parts of the gland and augment the evidence of the heterogeneity of the rat pinealocytes. It is possible that cholinergic neurons in the medial habenular nuclei or in the parasympathetic sphenopalatine ganglion may be a source of these AChE-positive fibres. The examination was performed at the light microscope level.     

Calcified inclusions in the superficial pineal gland of the mongolian gerbil, Meriones unguiculatus.

A histological and histochemical study of the pineal gland of neonatal, juvenile and adult gerbils is described. Calcified inclusions appear within pinealocytes in the superficial pineal about the third week of age, and the incidence of inclusions increased with age until, by the eleventh week, they are found in all animals. The inclusions contain an organic matrix composed of a carbohydrate, probably an acid mucopolysaccharide, complexed to protein. Calcification does not occur in the deep pineal. The data are interpreted to indicate that the formation of calcified inclusions is a normal process within the gerbil pineal. The similarity of the process of calcification in the gerbil and in the human pineal suggests that the gerbil may be an animal of choice for the controlled study of the phenomenon of pineal calcification.     

The expression of α-internexin and peripherin in the developing mouse pineal gland

Summary The mammalian pineal gland contains pinealocytes, interstitial glial cells, perivascular macrophages, neurons and neuron-like cells. The neuronal identity of neurons and neuron-like cells was an enigma. α-Internexin and peripherin are specific neuronal intermediate filament proteins and are expressed differentially in the CNS and PNS. We investigated the development of immunoreactivity and expression patterns of mRNAs for α-internexin and peripherin in the mouse pineal gland to determine the neuronal identity of these cells. Both α-internexin- and peripherin-immunoreactive cells were readily visualized only after birth. Both proteins were at the highest level on the postnatal day 7 (P7), rapidly declined at P14, and obtained their adult level at P21. Both protein and mRNA of α-internexin are expressed in some cells and nerve processes, but not all, of adult mouse pineal gland. Less number of peripherin immunoreactive or RNA-expressing cells and nerve processes were identified. Accumulations of α-internexin and peripherin proteins were also found in the cells from the aged pineal gland (P360). We concluded that some cells in the developing mouse pineal gland may differentiated into neurons and neuron-like cells expressing both α-internexin and/or peripherin only postnatally, and these cells possess dual properties of CNS and PNS neurons in nature. We suggested that they may act as interneurons between the pinealocyte and the distal neurons innervating the pinealocytes, or form a local circuitry with pinealocytes to play a role of paracrine regulatory function on the pinealocytes.     

Neuropeptide Y Inhibits β-Adrenergic Agonist– and Vasoactive Intestinal Peptide–Induced Cyclic AMP Accumulation in Rat Pinealocytes Through Pertussis Toxin–Sensitive G Protein

The effects of neuropeptide Y (NPY) on pineal gland cyclic AMP (cAMP) accumulation were investigated using dispersed pinealocytes from rats. NPY inhibited the intracellular cAMP accumulation stimulated by isoproterenol and norepinephrine in a dose-dependent manner during a 10-min incubation of pinealocytes. NPY (1 x 10(-7) M) also inhibited vasoactive intestinal peptide (VIP)- and cholera toxin-induced cAMP accumulation. The inhibitory effect of NPY on isoproterenol-induced cAMP accumulation was completely abolished by a 5-h pretreatment of pinealocytes with 1 microgram/ml of pertussis toxin (PT). These results suggest that NPY participates in modulation of cAMP production in the rat pineal gland through PT-sensitive G protein. Yohimbine, an alpha 2-adrenergic antagonist, blocked NPY inhibition of isoproterenol-stimulated cAMP accumulation. On the other hand, the alpha 2-adrenergic agonist clonidine by itself did not affect cAMP accumulation stimulated by isoproterenol but significantly potentiated NPY action. The present study demonstrates that NPY inhibits beta-adrenergic or VIPergic stimulation of the pineal gland cAMP accumulation. The inhibitory effect of NPY is mediated through PT-sensitive G protein. Our results also suggest that NPY exerts its action to affect alpha 2-adrenoceptor function.     

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.     

Expression of the Otx2 homeobox gene in the developing mammalian brain: embryonic and adult expression in the pineal gland

Otx2 is a vertebrate homeobox gene, which has been found to be essential for the development of rostral brain regions and appears to play a role in the development of retinal photoreceptor cells and pinealocytes. In this study, the temporal expression pattern of Otx2 was revealed in the rat brain, with special emphasis on the pineal gland throughout late embryonic and postnatal stages. Widespread high expression of Otx2 in the embryonic brain becomes progressively restricted in the adult to the pineal gland. Crx (cone-rod homeobox), a downstream target gene of Otx2, showed a pineal expression pattern similar to that of Otx2, although there was a distinct lag in time of onset. Otx2 protein was identified in pineal extracts and found to be localized in pinealocytes. Total pineal Otx2 mRNA did not show day-night variation, nor was it influenced by removal of the sympathetic input, indicating that the level of Otx2 mRNA appears to be independent of the photoneural input to the gland. Our results are consistent with the view that pineal expression of Otx2 is required for development and we hypothesize that it plays a role in the adult in controlling the expression of the cluster of genes associated with phototransduction and melatonin synthesis.     

Morphofunctional changes in the pineal gland during adaptation to hypothermia

The influence of hypothermal stress (+4 degrees during 3 h) on the ways of serotonin metabolism in pineal gland and its structure has been studied in dynamics on adult male Wistar rats. It has been revealed that melatonin-producing epiphyseal function suffers from phase changes in dynamics of adaptation--significant rising during 15 min. after beginning of the experiment, rehabilitation up to normal--in 30 min, and fast suppressing--in 3 hrs. Suppressing of the functional pineal activity is not due to switched serotonin metabolism with melatonin and new indoles release, but to a partial pinealocytes breaking from their active function.     

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.     

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.     

Synaptic ribbons during postnatal development of the pineal gland in the golden hamster (Mesocricetus auratus)

Summary Synaptic ribbons, functionally enigmatic structures of mammalian pinealocytes, were studied during the postnatal development of the pineal gland in the golden hamster (Mesocricetus auratus). On day 4 post partum, synaptic ribbons appear in cells that have already started to differentiate into pinealocytes. Between days 4 and 9, an increase in the number of synaptic ribbons occurs, concomitant with the continuing differentiation of the pineal tissue. Between days 9 and 16, when differentiation of this tissue is almost completed, the number of synaptic ribbons decreases and approaches that characteristic of the adult pineal gland. During development, the synaptic ribbons increase in length, and dense core vesicles are frequently found in the vicinity of these structures. It is assumed that a functional relationship exists between dense core vesicles and the synaptic ribbons, which are considered to be engaged in a certain form of secretory activity of the mammalian pineal gland.Supported by the Deutsche Forschungsgemeinschaft     

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.     

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.     

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.     

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.     

Uptake of estradiol by the rat pineal organ. Effects of cervical sympathectomy, stage of the estrous cycle and estradiol treatment

Rat pineal organs of spayed rats took up and retained estradiol $\text{in vitro}$ up to 32-fold the concentration present in the incubation media. This phenomenon was maximum at 37°C and after 2-h incubations. Most (86–91%) of [³H] radioactivity recovered from the incubated pineals was identified as estradiol by thin-layer chromatography. Treatment with dextran-coated charcoal of nuclei-free pineal homogenates incubated with [³H] estradiol of different SA uncovered a high affinity, low capacity binding of estradiol to cytosol components. Uptake of estradiol by the nuclear fraction also proceeded in a saturable fashion. Similar findings were made in uterine homogenates of spayed rats.Estradiol uptake by the pineal organ and the uterus of cycling rats varied as a function of the stage of the estrous cycle, maxima being observed in diestrus and minima in proestrus. The administration of a priming dose of estradiol benzoate to spayed rats caused high affinity binding components of the pineal cytosol to increase by about 150%. Nuclear binding of estradiol was also increased by the estradiol priming dose. Pineal denervation, i.e., by superior cervical ganglionectomy, caused pineal estradiol uptake to decrease significantly by about 20%. These data suggest that the early steps of estradiol action on the pineal organ may resemble those of the uterus.     

Neurokinin A in the anterior pituitary of female rats: effects of ovariectomy and estradiol.

The effect of acute and chronic ovariectomy and the substitutive treatment with 17-beta estradiol and/or progesterone on anterior pituitary levels of neurokinin A (NKA) was studied in female rats. Acute ovariectomy did not result in significant changes of NKA in the anterior pituitary gland as compared with the levels in diestrous intact rats, but a single injection of 5 micrograms of estradiol in ovariectomized rats significantly decreased NKA levels in the anterior pituitary gland. Progesterone was without effect and did not modify the decrease of NKA in the anterior pituitary gland induced by estradiol. In rats examined 11 to 17 days after ovariectomy, NKA in the anterior pituitary gland was significantly higher than in diestrous intact rats. In the hypothalamus, ovariectomy resulted in decreased levels of NKA in the median eminence-arcuate nucleus. Estradiol significantly reduced NKA stores in the anterior pituitary gland but increased them in the whole hypothalamus and in the median eminence-arcuate nucleus. Thus, estradiol seems to be a powerful regulator of NKA stores in the adenohypophysis and also in the hypothalamus.     

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.     

A Comparison of the Effects of Estrogen and Cimicifuga racemosa on the Lacrimal Gland and Submandibular Gland in Ovariectomized Rats

This study aims to observe the effects of estradiol and Cimicifuga racemosa on the lacrimal gland and submandibular gland of ovariectomized rats. We randomly divided 20 adult female SD rats into four groups—a sham-operated group (SHAM), ovariectomized (OVX) group, ovariectomized group treated with estradiol (OVX+ E), and ovariectomized group treated with the isopropanolic extract of Cimicifuga racemosa (OVX+ iCR). The SHAM group and OVX group used distilled water to instead the drugs. Two weeks after ovariectomy, the estradiol and iCR were administered for 4 weeks. Next, we used H&E staining and electron microscopy to observe any histological changes in the lacrimal and submandibular glands and immunohistochemical staining to observe the expressions of cleaved caspase-3 (Casp-3) and Cu-Zn SOD (superoxide dismutase). The H&E staining find that both drugs can prevent the cells of area from shrinkage in the two kinds of gland. But under the electron microscopy, estradiol and iCR have different efficacy. Estradiol is more effective at protecting mitochondria in lacrimal gland acinar cells than iCR, and iCR is more effective at suppressing endoplasmic reticulum expansion than estradiol. Both estradiol and iCR have a similar protective function on mitochondria in the submandibular gland. The protective function of the two glands may inhibit apoptosis by suppressing the expression of Casp-3. In addition, iCR increases the expression of Cu-Zn SOD in duct system of submandibular gland. The results suggest that both estradiol and iCR confer a protective effect on the lacrimal and submandibular glands of ovariectomized rats via different mechanisms.     

Influence of 4-day long treatment with vasoactive intestinal peptide on ultrastructure and function of the rat pinealocytes in organ culture

Vasoactive intestinal peptide (VIP) is one of neuropeptides involved in the regulation of the pineal gland function. The acute treatment of rat pinealocytes with VIP caused changes in their biochemical parameters. The present study concerns the effects of the chronic treatment with VIP on ultrastructure and function of the rat pinealocytes in organ culture. The pineals of adult male rats were assigned to one of three groups and placed in organ culture for four consecutive days. The pineals of the first group were incubated in the control medium, the pineals of the second group--12 hrs in control medium and 12 hrs in medium with 1 microM VIP (between 20.00 and 8.00) during each day, the pineals of the third group--24 hrs per day in medium with 1 microM VIP. The melatonin concentration was measured using RIA and activity of enzymes using radiochemical methods. Point count method was used in quantitative ultrastructural analysis. Both modes of chronic treatment with VIP increased significantly the level of melatonin secretion during four days of the culture and the content of this hormone in the pineal explants at the end of the experiment. Treatment with the neuropeptide for 12 hrs and 24 hrs per day elevated also the activity of arylalkylamine N-acetyltransferase and hydroxyindole-O-methyltransferase. On the other hand, VIP had no effect on the activity of arylamine-N-acetyltransferase. VIP increased the relative volume of rough endoplasmic reticulum, Golgi apparatus and mitochondria and did not influence the relative volume of lysosomes and lipid droplets as well as the numerical density of dense core vesicles in the examined rat pinealocytes. The obtained results indicate stimulatory effect of chronic treatment with VIP on the synthesis and secretion of melatonin in the rat pinealocytes in vitro. The results of morphological study are in agreement with the obtained biochemical data and point to the increase in secretory and metabolic activity of the rat pinealocytes in response to VIP.     

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.