Innervation of the pineal gland in the Arctic fox (Vulpes lagopus) by nerve fibres immunoreactive to substance P and calcitonin gene-related peptide.

BACKGROUND: The study demonstrates, for the first time, the presence of substance P (SP) and calcitonin gene-related peptide (CGRP) in the nerve fibres supplying the pineal gland in the Arctic fox. MATERIALS AND METHODS: The expression and distribution pattern of the studied substances were examined by double-labelling immunofluorescence technique. RESULTS: The SP-positive fibres enter into the pineal gland through the capsule as the nervi conarii. The fibres formed thick bundles in the capsule and connective tissue septa, from where they penetrated into the pineal parenchyma. Inside the parenchyma, the nerve fibres created basket-like structures surrounding clusters of pinealocytes. The density of intrapineal SP positive fibres was slightly higher in the distal and middle parts of the gland than in the proximal one. Double immunostaining with antibodies against SP and CGRP revealed that the vast majority of SP positive fibres were also CGRP positive. The fibres showing a positive reaction to SP and negative to CGRP were scattered within the whole gland. The fibres immunopositive to CGRP and immunonegative to SP were not observed. In the habenular and posterior commissural areas adjoining to the pineal gland the immunoreactive nerve fibres were not found. Moreover, no immunopositive cell bodies were observed in both the pineal gland and the commissural areas. CONCLUSIONS: These results reveal that SP and CGRP are involved in the innervation of pineal gland in carnivores. In turn we suggest that these peptides can regulate/modulate melatonin secretion.

Diurnal and circadian variations in indole contents in the goose pineal gland.

The diurnal and circadian profiles of pineal indoles, except melatonin, are poorly characterized in birds. Moreover, there are no data on the effect of sudden changes in the light-dark cycle on these profiles. Therefore, we investigated the diurnal (Experiment I) and circadian variation (Experiment II) of nine pineal indoles (tryptophan, 5-hydroxytryptophan, serotonin, N-acetylserotonin, melatonin, 5-hydroxyindole acetic acid, 5-methoxytryptophol, 5-methoxyindole acetic acid, 5-methoxytryptamine) in geese, as well as the changes in the profiles of these substances in geese subjected to a reversed light-dark cycle (Experiment III). For the first 12 weeks of life, all geese were kept under a diurnal cycle of 12 h of light and 12 h of darkness (12L:12D). In Experiment I (n = 48), they were kept under these conditions for another 14 days before being sacrificed at 2-h intervals for sampling of the pineal glands. In Experiment II, the geese (n = 48) were divided into three groups (12L:12D, 24L:0D, 0L:24D) for 10 days before sampling at 6-h intervals. In Experiment III, 24 geese were exposed to a reversed light-dark cycle before sampling at 14:00 and 02:00 on the first, second and third days after light-dark cycle reversal. To determine the content of the indoles in the goose pineals, HPLC with fluorescence detection was used. We found that, with the exception of tryptophan, all the investigated indoles showed statistically significant diurnal variation. When geese were kept in constant darkness, most of the indoles continued to show this variation, but when geese were kept in constant light, the indoles did not show significant variation. When the light-dark cycle was reversed (12L:12D to 12D:12L), the profiles of NAS, melatonin, 5-MTAM and 5-MTOL reflected the new cycle within 2 days. The content of serotonin in geese in 12L:12D was higher than that observed in other birds under these conditions, which suggests that this compound may play a special role in the pineal physiology of this species. In conclusion, our results show that the daily variations in the metabolism of melatonin-synthesis-related indoles in the goose pineal gland are generated endogenously and controlled by environmental light conditions, as in other birds. However, comparison of the results obtained with the goose to those obtained with other species (chicken, duck) unambiguously shows that the profiles of pineal indoles differ markedly between species, in both the quantitative proportions of the compounds and the characteristics of the diurnal changes. These findings provide strong arguments for the need for comparative studies.

Adrenergic control of pinealocyte chondriome - an in vitro study.

Norepinephrine released from sympathetic innervation plays the main role in the regulation of melatonin secretion in mammalian pinealocytes. The present study was conducted for the following reasons: 1) to establish whether the pinealocyte chondriome is controlled by norepinephrine, 2) to determine the effect of adrenergic stimulation on mitochondria, and 3) to characterize adrenoceptors involved in the regulation of the chondriome. The static organ culture of the pineal gland was used. The explants were incubated for 5 consecutive days in control medium and between 20:00 and 08:00 in medium with the presence of 10 µM norepinephrine - adrenergic agonist; isoproterenol - beta-adrenoceptor agonist; cirazoline, methoxamine, M-6364 - alfa1 - adrenoceptors agonists or PMA - activator of PKC. The explants were then subjected to ultrastructural examination and morphometric analysis. The incubation of explants in the presence of norepinephrine or isoproterenol caused a decrease in the relative volume and the numerical density of mitochondria and induced an increase in the percentage of free mitochondria in pinealocytes. Significant changes in these parameters were not observed after treatment with methoxamine, cirazoline, M-6463 and PMA. The results obtained show that the chondriome of pig pinealocytes is controlled by norepinephrine acting via beta-adrenoceptors. Adrenergic stimulation, repeated for five consecutive days of organ culture, causes a decrease in the number of mitochondria and a shift in the distribution of mitochondria from the form of networks and filaments into the form of single particles. This indicates the intensive remodeling of the mitochondria network, which is closely linked to the metabolic status of the cell.

Regulation of melatonin secretion in the pineal organ of the domestic duck--an in vitro study.

The aim of study was to determine the mechanisms regulating melatonin secretion in the pineal organs of 1-day-old and 9-month-old domestic ducks. The pineals were cultured in a superfusion system under different light conditions. Additionally, some explants were treated with norepinephrine. The pineal glands of 1-day-old ducks released melatonin in a well-entrained, regular rhythm during incubation under a 12 hrs light:12 hrs dark cycle and adjusted their secretory activity to a reversed 12 hrs dark:12 hrs light cycle within 2 days. In contrast, the diurnal changes in melatonin secretion from the pineals of 9-month-old ducks were largely irregular and the adaptation to a reversed cycle lasted 3 days. The pineal organs of nestling and adult ducks incubated in a continuous light or darkness secreted melatonin in a circadian rhythm. The treatment with norepinephrine during photophases of a light-dark cycle resulted in: 1) a precise adjustment of melatonin secretion rhythm to the presence of this catecholamine in the culture medium, 2) a very high amplitude of the rhythm, 3) a rapid adaptation of the pineal secretory activity to a reversed light-dark cycle. The effects of norepinephrine were similar in the pineal organs of nestlings and adults. In conclusion, melatonin secretion in the duck pineal organ is controlled by three main mechanisms: the direct photoreception, the endogenous generator and the noradrenergic transmission. The efficiency of intra-pineal, photosensitivity-based regulatory mechanism is markedly lower in adult than in nestling individuals.

Neuropeptide Y as a presynaptic modulator of norepinephrine release from the sympathetic nerve fibers in the pig pineal gland.

Norepinephrine (NE) released from the sympathetic nerve endings is the main neurotransmitter controlling melatonin synthesis in the mammalian pineal gland. Although neuropeptide Y (NPY) co-exists with NE in the pineal sympathetic nerve fibers it also occurs in a population of non-adrenergic nerve fibers located in this gland. The role of NPY in pineal physiology is still enigmatic. The present study characterizes the effect of NPY on the depolarization-evoked 3H-NE release from the pig pineal explants. The explants of the pig pineal gland were loaded with 3H-NE in the presence of pargyline and superfused with Tyrode medium. They were exposed twice to the modified Tyrode medium containing 60 mM of K+ to evoke the 3H-NE release via depolarization. NPY, specific agonists of Y1- and Y2- receptors and pharmacologically active ligands of α2-adrenoceptors were added to the medium before and during the second depolarization. The radioactivity was measured in medium fractions collected every 2 minutes during the superfusion. NPY (0.1-10 µM) significantly decreased the depolarization-induced 3H-NE release. Similar effect was observed after the treatment with Y2-agonist: NPY13-36, but not with Y1-agonist: [Leu31,Pro34]-NPY. The tritium overflow was lower in the explants exposed to the 5 µM NPY and 1 µM rauwolscine than to rauwolscine only. The effects of 5 µM NPY and 0.05 µM UK 14,304 on the depolarization-evoked 3H-NE release were additive. The results show that NPY is involved in the regulation of NE release from the sympathetic terminals in the pig pineal gland, inhibiting this process via Y2-receptors.

In vitro effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on ovarian, pituitary, and pineal function in pigs.

The aims of the study were: (1) to examine 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and/or prolactin (PRL) effects on in vitro secretion of progesterone (P(4)) and estradiol (E(2)) by luteinized granulosa and theca cells from porcine preovulatory follicles; and (2) to determine the effects of TCDD on PRL, luteinizing hormone (LH), and melatonin luteal phase in pigs. We found that TCDD itself did not affect progesterone secretion, but it abolished the stimulatory effect of PRL in the follicular cells. TCDD stimulated PRL secretion during the luteal phase and inhibited during the follicular phase. Moreover, TCDD increased luteinizing hormone secretion by pituitary cells during the follicular phase. In contrast to protein and steroid hormones, melatonin secretion in vitro was not affected by TCDD. In conclusion, it was found that the pituitary-ovarian axis in pigs is sensitive to TCDD, and the dioxin exhibited a profound ability to disrupt the ovarian actions of prolactin.

The influence of methisoprinol administered in ovo on the morphological structure of the spleen in turkeys.

This study analyzed the effect of a synthetic, low-toxic immunomodulator - methisoprinol - administered in ovo on the morphological structure of the spleen in turkeys. Experiments were conducted on three groups of 5-day-old BUT 9 turkeys (35 birds in each group) hatched from eggs which, on day 26 of incubation, had been administered methisoprinol (VetAgro, Lublin, Poland) in ovo in a dose of 5 mg (group I) or 20 mg per egg (group II). Poults hatched from eggs administered a physiological solution of NaCl in a dose of 0.1 ml per egg in ovo served as a control (group III). Samples of the spleen were collected from 5 birds selected at random from a group of decapitated 5-day-old poults and the prepared 7 fm-paraffin sections were stained with HE. A morphometric analysis of the germinal centres of the white pulp of the spleen was conducted by subjecting pictures taken with an optical microscope to a Digital Image Analysis using Axio Vision software (by Zeiss). The study demonstrated that in terms of the morphological structure, the spleen of the poults hatched from eggs administered 5 mg of methisoprinol (group I) did not differ considerably from the spleen of the control birds. In turn, spleens of the poults hatched from eggs administered 20 mg of methisoprinol per embryo were characterized by distinctively developed red pulp and within the area of the white pulp by distinct cortical section containing numerous lymphocytes. In spleens of the poults from this group, the morphometric examination also demonstrated a higher number of germinal centres of the white pulp as compared to their number in spleens of the birds from the other groups.

Calcium concrements in the pineal gland of the Arctic fox (Vulpes lagopus) and their relationship to pinealocytes, glial cells and type I and III collagen fibers.

The aim of the present study was to analyze the presence and morphology of the pineal concretions in the Arctic fox and their relationship to pinealocytes, glial cells and collagen fibers. Pineals collected from 7-8 month-old and 3-4 year-old foxes (6 in each age-group) were investigated. Sections of the glands were stained with HE, Mallory's method and alizarin red S as well as subjected to a combined procedure involving immunofluorescent staining with antibodies against antigen S, glial fibril acid protein (GFAP), type I and III collagen and histochemical staining with alizarin red S. The pineal concretions were found in 2 of 6 investigated Arctic foxes aged 3 years and they were not observed in animals aged 7-8 months. The acervuli were present in the parenchyma and the connective tissue septa. They were more numerous in the distal part than in the proximal part of the gland. The acervuli stained with alizarin red S revealed an intensive red fluorescence, what enabled the use of this compound in a combined histochemical-immunofluorescent procedure. A majority of cells in the fox pineal showed positive staining with antibodies against antigen S, a marker of pinealocytes. GFAP-positive cells were especially numerous in the proximal part of the gland. Both antigen S- and GFAP-positive cells were frequently observed close to the concrements. Collagen fibers of type I and III were found in the capsule, connective tissue septa and vessels. Immunoreactive fibers did not form any capsules or basket-like structures surrounding the concrements.

Pineal concretions in turkey (Meleagris gallopavo) as a result of collagen-mediated calcification.

The intra-pineal calcification is a well-known phenomenon in mammals, however it is almost completely unknown in birds. The aim of the present work was to analyze morphology and genesis of the pineal concretions in the turkey. The studies were performed on the pineals collected from one-year-old turkeys (Meleagris gallopavo). In addition to standard morphological methods, the alizarin red S and potassium pyroantimonate methods were employed for localization of calcium at the light and electron microscopy level. In light microscopy, calcified concretions with diameters from 300 microm to 2 mm and quantities from 3 to 6 per gland were observed in all the examined pineals. They were stained red with alizarin S and showed the presence of collagen in Mallory's staining. Two types of cells were noted inside the concretion: polygonal and elongated ones. Using electron microscopy, three parts were distinguished within the calcification area. The peripheral part contained densely packed collagen fibrils, some elongated cells and numerous pyroantimonate precipitates demonstrating the presence of calcium ions. In the intermediate part, the fibrils were covered by almost continuous sheets of pyroantimonate precipitates and fused side by side. The central part showed an appearance of calcified hard tissue and contained some polygonal (osteocyte-like) cells. The obtained data demonstrated that the formation of the pineal concretions in the turkey is associated with the mineralization of collagen. This process is completely different from the mechanisms responsible for the formation of the concretions in the mammalian pineal.

Peptidergic and nitrergic innervation of the pineal gland in the domestic pig: an immunohistochemical study.

The presence and co-localization of vasoactive intestinal polypeptide (VIP), peptide N-terminal histidine C-terminal isoleucine (PHI), pituitary adenylate cyclase-activating peptide (PACAP), somatostatin (SOM), calcitonin gene-related peptide (CGRP), substance P (SP) and the neuronal isoform of nitric oxide synthase (NOS) were studied in neuronal structures of the pig pineal gland. Paraformaldehyde-fixed pineals of 3-month-old gilts were sliced into serial cryostat sections, which were subjected to a set of double immunofluorescence stainings. Based on the co-existence patterns of neuropeptides, five populations of nerve fibres supplying the pig pineal were distinguished: (1) PHI-positive, (2) PACAP-positive, (3) SOM-positive, (4) SP/CGRP-positive and (5) SP-positive/CGRP-negative. Only a subpopulation of PHI-positive fibres contained VIP at the level detectable by immunofluorescence. NOS was found in some intrapineal PHI- and VIP-positive fibres. PHI-, VIP- and NOS-positive nerve fibres were more numerous in the peripheral than in the central part of the pineal. PACAP-positive fibres were equally distributed within the gland. The density of SOM-positive fibres was higher in the ventro-proximal than in the dorso-distal part of the pineal. SOM was also detected in some neuronal-like cells or specialized pinealocytes situated in the central region of the gland. Two populations of fibres containing SP were found: CGRP-positive, present in the distal and central parts of the pineal as well as CGRP-negative, localized in the proximal compartment of the gland.

Histology and ultrastructure of the pineal organ in the domestic goose.

The pineal organs of 14-week-old domestic geese were investigated with light and electron microscopy. The pineals consisted of a wide distal part and a narrow middle-proximal one. The glands were attached to the intercommissural region via the choroid plexus. The pineal parenchyma was formed by round or elongated follicles. The follicular wall was composed predominantly by cells immunoreactive with antibodies against hydroxyindolo-O-methyltransferase (HIOMT) or glial fibrillary acid protein (GFAP). They formed two or more layers. HIOMT-positive elements were represented by elongated cells bordering the follicular lumen and oval cells located in the external layer of the follicular wall. These cells were identified in ultrastructural studies as rudimentary-receptor pinealocytes and secretory pinealocytes, respectively. Among rudimentary-receptor pinealocytes two types of cells, designed as A and B, were distinguished due to structural differences. Type A cells extended through the whole follicular wall and showed regular stratified distribution of organelles in well-recognizable zones with rough endoplasmic reticulum, the Golgi apparatus and mitochondria. Type B cells, like type A pinealocytes, contacted the pineal lumen and showed polarity of their internal structure. However, they were markedly shorter than the cells of type A and lacked stratified distribution of organelles. Secretory pinealocytes contained irregularly dispersed organelles. A prominent feature of all types of goose pinealocytes was the presence of numerous dense core vesicles. The population of GFAP-positive cells consisted of ependymal-like supporting cells and astrocyte-like cells.

Distribution of NADPH-diaphorase activity in the pineal gland of the Turkey.

NADPH-diaphorase activity was histochemically demonstrated in the nerve fibers, neuronal-like cell bodies and in the endothelial cells of the vasculature in the pineal gland of the turkey. The nerve fibers were localized in the choroid plexus, connecting the pineal gland with the diencephalon as well as inside the pineal gland, where they formed basket-like structures around the pineal follicles. A group of neuronal-like cell bodies was observed in the proximal part of the gland. The positive staining was not observed in the pinealocytes of rudimentary-photoreceptor type and in the supporting cells.

Leu-enkephalin immunoreactivity in the pig pineal gland.

Leu-enkephalin-positive structures in the pig pineal gland were demonstrated immunohistochemically using mouse monoclonal antibody. The pineal glands were obtained from the newborn, 21-day and 7-month old female pig. The immunopositive nerve fibers were observed in the pineal gland as well as in the epithalamic areas. The leu-enkephalin-immunoreactive nerve fibers (single or forming small bundles) were localized mainly in the proximal part of the pineal and they were scarce in other parts. The localization of the fibers points to a central source of this innervation. The study did not show any age-dependent differences in the distribution and density of leu-ekephalin-positive nerve fibers.

Pituitary adenylate cyclase-activating polypeptide-immunoreactive (PACAP-IR) nerve fibers in the pig pineal gland.

The present study demonstrates the occurrence of PACAP-immunoreactive (PACAP-IR) nerve fibers in different compartments of the pig pineal gland, including glandular capsule (where they form a very dense network) and subependymal tissue close to the pineal recess (moderate to dense meshwork of varicose fibers). Furthermore, several varicose fibers penetrate from the capsule into the connective tissue septa and then into the parenchyma, where they form unequally distributed, fine network and, in some cases, basket-like structures around pinealocytes. Some of the PACAP-IR nerve fibers, observed both in the habenular and posterior epithalamic areas, extend to the pineal gland. PACAP-IR cells could be demonstrated neither in the pineal gland, nor in epithalamic areas.

Localization of NADPH-diaphorase activity in the pineal gland of the domestic pig.

The study showed the presence of NADPH-diaphorase containing structures in the pineal gland of the domestic pig. NADPH-diaphorase activity was found in the nerve fibers and in the endothelial cells of the vasculature. The nerve fibers were localized in the capsule, around the blood vessels as well as in the parenchyma. The positive staining was not observed in the pig pinealocytes.

Distribution of growth associated protein (GAP-43) immunoreactivity in nerve fibers supplying the pig pineal gland.

An immunohistochemical study of the pig pineal gland was carried out using monoclonal mouse antiserum against growth-associated protein GAP-43. The pineal glands were obtained from the 3, 5, 8 weeks old piglets. The immunopositive nerve fibers were observed in the pineal gland as well as in the habenular and the posterior comissural areas. They formed a dense network in the habenular area and the proximal part of the pineal gland. In the comissural area and in the apical part of the gland. single positive fibers were observed. The obtained results may suggest a difference in the plasticity of innervation between the particular regions of the pineal gland.

Immunohistochemical localization of substance P in the pineal gland of the domestic pig.

An immunohistochemical study of the pig pineal gland was carried out using polyclonal rabbit antiserum raised against substance P (SP). The pineal glands were taken from the newborn, 21-day- and 7-month-old female pigs. Immunoreactive nerve fibers were observed in the pineal gland as well as in the posterior commissure and habenular areas. The bundles of SP-immunoreactive fibers were also seen in the subependymal layer of the pineal tissue. The single SP-immunoreactive nerve fibers and few small bundles of nerve fibers were located with equal density throughout the pineal gland, in the connective tissue septa and in the parenchyma. SP-immunoreactive cell bodies were observed in the medial habenular nucleus. The obtained results point to this nucleus as one of the central sources of SP innervation in the pig pineal gland. The study did not show any differences in the distribution and the density of SP-immunoreactive nerve fibers between newborn, 21-day- and 7 month-old pigs.

Somatostatin and somatostatin receptors in the pig pineal gland during postnatal development: an immunocytochemical study.

An immunohistochemical study of the pineal gland of the domestic pig was carried out using rabbit antisera raised against synthetic peptide fragments corresponding to different amino acid sequences of the prosomatostatin, the somatostatin-14, and the somatostatin-28 molecule. The study was supplemented by immunohistochemical staining with rabbit antisera raised against five subtypes of somatostatin receptors. The pineal glands were taken from the newborn, 21-day-old and 7-month-old pigs. Immunoreactive nerve fibers and cells were observed in the pineal gland with all the antisera against somatostatin and prosomatostatin. The nerve fibers were located throughout the pineal gland-in the capsule, connective septa, and parenchyma-with the highest density in proximo-ventral part of the gland. The somatostatin positive fibers were also found in the habenular and posterior commissurae areas. Somatostatin-immunoreactive cell bodies were observed mostly in the central part of the gland. These results point to the existence of two somatostatin sources in the pig pineal gland: 1) nerve fibers, probably of central origin; and 2) cells that may represent intrapineal neurons or specialised pinealocytes. A clear difference in the immunoreactivity between newborn, 21-day-old, and 7-month-old pigs was found. Generally, the density of nerve fibers was lower in adult than young animals. The number of the cells also decreased with age. By using the antisera against the five somatostatin receptors, only sst3 - receptor immunoreactivity could be detected. The receptor-immunoreactivity was confined to varicose and smooth fibers and some cells. The sst(3)-receptor positive structures were localised in all parts of the gland and their number was higher in younger pigs.