Neuronal markers in the rodent pineal gland — an immunohistochemical investigation

Summary 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 immunfluorescence. 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 situhybridisation of relevant mRNAs will be necessary to answer these questions definitely.The data presented in this study form part of a thesis presented by A. Bendig in partial fulfillment for the degree of M.D..Supported by the Deutsche Forschungsgemeinschaft, grant Schr 283/2-1     

Immunocytochemical demonstration of rod-opsin,S-antigen,and neuron-specific proteins in the human pineal gland

Summary The aim of this study was to examine whether rod-opsin and S-antigen immunoreactions were present in the pineal organ of adult man and how these immunoreactions were correlated with neuronal markers, e.g., synaptophysin, and neurofilaments L, H and M. Three perfusion-fixed epithalamic regions including the pineal organ and five pineal glands obtained at routine autopsy were used. The specimens were taken from female or male patients, 25 to 85 years of age. All immunoreactions were performed using highly specific, well-characterized antibodies. Rod-opsin and S-antigen-immunoreactive pinealocytes occurred in all pineal organs investigated; however, the immunoreaction was restricted to small subpopulations of pinealocytes (rod-opsin immunoreaction: approximately 3%–5%; S-antigen immunoreaction: approximately 5%–10% of the total population). In contrast, immunoreactions for synaptophysin and neurofilaments M and H were present in numerous pinealocytes. Immunoreactivity for neurofilament L was not found. These data suggest that the cellular composition of the human pineal organ is heterogeneous. Moreover, the presence of rod-opsin and S-antigen immunoreactions in the human pineal organ indicates that it may be affected by autoimmune retinal diseases that are provoked by antibodies against these proteins, as is the case in rodents and non-human primates.     

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.     

Neuropeptide Y (NPY)-like immunoreactivity in peripheral and central nerve fibres of the golden hamster (Mesocricetus auratus) with special respect to pineal gland innervation

Summary Inforimation on the ambient lighting conditions is conveyed from the retina to the pineal organ by a neuronal pathway involving the suprachiasmatic nucleus (SCN) which acts as a circadian pacemaker. In the hamster, circadian rhythms have been shown to be influenced by injection of neuropeptide Y (NPY) into the SCN. Since NPY-immunoreactive nerve fibres are present in the rat and guinea-pig pineal glands it appeared of interest to investigate the hamster pineal as part of the circadian rhythm generating/regulating system. For comparison kidney, small intestine and cerebral cortex were studied. Like in the other rodent species so far investigated only a few of the abundant sympathetic nerve fibres in the hamster pineal gland are NPY-immunoreactive, in contrast to the relatively rich innervation of the other organs. This speaks in favour of a possible central origin of pineal NPY-immunoreactive fibres. These may either exert vasoregulatory effects on pineal vasculature or be involved in the modulation of alpha-adrenergic receptor mediated regulation of pineal metabolism.Supported by the Deutsche Forschungsgemeinschaft, grant Schr 283/1-1     

Neuropeptide Y (NPY)-like immunoreactivity in peripheral and central nerve fibres of the golden hamster (Mesocricetus auratus) with special respect to pineal gland innervation

Information on the ambient lighting conditions is conveyed from the retina to the pineal organ by a neuronal pathway involving the suprachiasmatic nucleus (SCN) which acts as a circadian pacemaker. In the hamster, circadian rhythms have been shown to be influenced by injection of neuropeptide Y (NPY) into the SCN. Since NPY-immunoreactive nerve fibres are present in the rat and guinea-pig pineal glands it appeared of interest to investigate the hamster pineal as part of the circadian rhythm generating/regulating system. For comparison kidney, small intestine and cerebral cortex were studied. Like in the other rodent species so far investigated only a few of the abundant sympathetic nerve fibres in the hamster pineal gland are NPY-immunoreactive, in contrast to the relatively rich innervation of the other organs. This speaks in favour of a possible central origin of pineal NPY-immunoreactive fibres. These may either exert vasoregulatory effects on pineal vasculature or be involved in the modulation of alpha-adrenergic receptor mediated regulation of pineal metabolism.     

Characteristic pattern of monoaminergic nerve fibers in the pineal organ of the monkey,Macaca fuscata

Summary Monoaminergic nerve fibers were studied in the pineal organ of the monkey, Macaca fuscata, by use of fluorescence and immunohistochemical procedures. Abundant formations of noradrenergic nerve fibers were observed in the pineal organ. They entered the parenchyma in the form of several coarse bundles via the capsule in the distal portion of the organ and spread throughout the organ after branching into smaller units. The density of the autonomic innervation decreased gradually toward the proximal portion of the organ. In the distal portion, numerous nerve fibers formed perivascular plexuses around the blood vessels and some fibers ran as bundles unrelated to the blood vessels in the stroma. Fine varicose fibers and bundles derived from these plexuses penetrated among the pinealocytes. However, only a few intraparenchymal fluorescent fibers were detected in the proximal third of the gland. With the use of serotonin antiserum serotonin-immunoreactive nerve fibers were clearly restricted to the ventroproximal part of the pineal organ. Although the somata of the pinealocytes showed intense immunoreactivity, their processes were not stained. In one exceptional case, clusters of pinealocytes displaying very intense immunoreactivity were found in an area extending from the distal margin of the ventral portion of the pineal stalk to the proximal portion of the pineal organ proper; these cells were bipolar or multipolar and endowed with well-stained processes.     

Demonstration of nerve fibers immunoreactive to peptide N-terminal histidine C-terminal isoleucine (PHI) and vasoactive intestinal peptide (VIP) in the pig pineal gland

The pineal functions are modulated by some neuropeptides including PHI and VIP. The presence of PHI-immunoreactive and VIP-immunoreactive nerve fibers in the pineal gland has been shown in several mammalian species. Both peptides influence the pineal serotonin N-acetyltransferase activity and melatonin synthesis. The aim of the present study was to examine the localization of PHI- and VIP-immunoreactive nerve fibers in the pig pineal gland. Four three-month old female pigs housed in natural light conditions, with free access to food and water, were used in the study. The pineals were fixed by perfusion with 4% paraformaldehyde in 0.1 M phosphate buffer. An immunohistochemical ABC streptavidin-biotin-complex method was used for the demonstration of PHI and VIP. PHI- and VIP-immunopositive nerve fibers were found in the pineal gland as well as in the habenular and posterior commissural areas. In the pineal gland, the density of PHI-immunoreactive nerve fibers was considerably higher than that of the fibers containing VIP. PHI- and VIP-immunopositive nerve fibers were more abundant in the cortical than in the medullary part of the gland. The nerve fibers formed bundles in the pineal capsule, from where they penetrated to the connective tissue septa and formed a dense meshwork surrounding blood vessels. In the parenchyma, PHI- and VIP-immunoreactive nerve terminals created baskets around clusters of pinealocytes. No PHI- or VIP-immunopositive cells were found in the pig pineal gland.     

Protein gene product (PGP) 9.5 immunoreactivity in nerve fibres and pinealocytes of guinea-pig pineal gland: interrelationship with tyrosine-hydroxylase- and neuropeptide-Y-immunoreactive nerve fibres

This light-microscopic (LM) immunohistochemical study has evaluated the presence and distribution of the pan-neural and neuroendocrine marker protein gene product (PGP) 9.5 in pinealocytes and nerve fibres of guinea-pig pineal gland. The pattern of PGP 9.5-immunoreactive (ir) nerve fibres has been compared with that of fibres staining for tyrosine hydroxylase (TH) or neuropeptide Y (NPY). The vast majority of pinealocytes stained for PGP 9.5, although with variable intensity. PGP 9.5 immunoreactivity was localized in pinealocytic cell bodies and processes. Double-immunofluorescence revealed that PGP 9.5 immunoreactivity was absent from glial cells identified with a monoclonal antibody against glial fibrillary acidic protein (GFAP), PGP 9.5 immunoreactivity was also present in a large number of nerve fibres and varicosities distributed throughout the pineal gland. The number of TH-ir and NPY-ir nerve fibres was lower compared with those containing PGP 9.5 immunoreactivity. All fibres staining for NPY also stained for TH. NPY-ir nerve fibres were found to be much more numerous than previously reported for this species. The double-immunofluorescence analysis indicated that almost all TH-ir nerve fibres of the pineal gland contained PGP 9.5 immunoreactivity. However, few PGP 9.5-ir nerve fibres, located in the periphery and the central part of the gland, were TH-negative. A large number of PGP 9.5-ir fibres was concentrated in the pineal stalk. In contrast, TH-ir and NPY-ir nerve fibres were rare in this part of the pineal gland. Our data provide evidence that immunohistochemistry for PGP 9.5 may be a useful tool further to differentiate central and peripheral origins of pineal innervation. Furthermore, the staining of pinealocytes for PGP 9.5 may be exploited to study the three-dimensional morphology and the architecture of pinealocytes and their processes under various experimental conditions.     

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.     

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.     

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.     

Cytochemical studies on cytoplasmic granular elements in the hamster pineal gland.

The pineal gland of adult golden hamsters (Mesocricetus auratus) was studied by various cytochemical methods at the electron microscopic level: (1) the modified chromaffin reaction specific for 5-hydroxytryptamine (5-HT), (2) argentaffin reaction, (3) zinc-iodide-osmium (ZIO) mixture reaction and (4) acid phosphatase reaction. In the pinealocytes, the dense-cored vesicles (80-160 nm in diameter) show both chromaffinity and argentaffinity, while the population of dense bodies (150-400 nm in diameter) is reactive to ammoniacal silver solution and ZIO mixture but not to the modified chromaffin reaction. After incubation for demonstration of acid phosphatase activity, reaction products are localized in some, but not all, of the dense bodies, in some of the small vesicles in the Golgi region and in one or two inner Golgi saccules. In nerve fibers in the pineal gland, small granulated vesicles are also reactive to the modified chromaffin reaction and ZIO mixture. Based upon these cytochemical results the following conclusions have been reached: (1) dense cored vesicles in the pinealocytes and small granulated vesicles in the nerve fibers of the hamster pineal gland contain 5-HT, and (2) the population of dense bodies in the pinealocytes is heterogeneous, some are lysosomes and the other are possibly the granules responsible for the secretion of pineal peptides.     

Innervation of the mink pineal gland with neuropeptide Y (NPY)-containing nerve fibers

Summary An immunohistochemical investigation of the mink pineal gland was performed by use of antibodies raised in rabbits against neuropeptide Y (NPY) and Cys-NPY (32–36)-amide recognizing neuropeptide Y with an amidation at position 36 (NPYamide). NPY-immunoreactive nerve fibers were located predominantly in the rostral part of the pineal gland and in the pineal stalk. Immunoreactive nerve fibers were found throughout the pineal gland, but the number of fibers in the caudal part of the gland was low. The fibers were present both in the perivascular spaces and between the pinealocytes. Many NPY-immunoreactive fibers were also located in the posterior and habenular commissures; some of these fibers were connected with the fibers in the rostral part of the mink pineal gland, indicating that at least some of the NPY-immunoreactive nerve fibers are of central origin. The nerve fibers immunoreactive to amidated NPY were distributed in a similar manner. However, the number of fibers immunoreactive to NPYamide was lower than the number of fibers immunoreactive to NPY itself. After removal of the superior cervical ganglia bilaterally 22 days or 12 months before sacrifice, NPY-immunoreactive nerve fibers remained in the gland. This immunohistochemical study of the mink pineal gland therefore shows that the NPY/NPYamide-immunoreactive nerve fibers innervating the pineal gland in this spegcies are a component of the central innervation or originnate from extracerebral parasympathetic ganglia.     

Photoreceptor-like outer segments in the pineal organ of the lovebird,Uroloncha domestica (Aves: Passeriformes)

Summary In the pineal organ of the lovebird, Uroloncha domestica, bulbous, cup-shaped and elongated outer segments of photoreceptor-like pinealocytes are demonstrated by scanning electron microscopy. These scarce outer segments, 4–11 m in length, extend into the pineal lumen. The present structural observations speak in favor of photosensitive pinealocytes in the pineal organ of Uroloncha domestica. The relation of the photoreceptor-like pinealocytes to acetylcholinesterase-positive nerve cells and a nervous connection between the pineal and the brain indicate that the pineal organ of this passeriform species may be the site of neuroendocrine and photoreceptive functions.Supported by a fellowship from the Japan Society for the Promotion of Science to M. UeckSupported by a grant from the Ministry of Education of Japan to K. Wake and by a grant of the Deutsche Forschungsgemeinschaft to M. Ueck     

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.     

Innervation of the cat pineal gland by neuropeptide Y-immunoreactive nerve fibers: an experimental immunohistochemical study

An immunohistochemical study of the cat pineal gland was performed using a rabbit polyclonal antibody directed against neuropeptide Y (NPY) and an antibody directed against the C-terminal flanking peptide of neuropeptide Y (CPON). Numerous NPY- and CPON-immunoreactive (IR) nerve fibers were demonstrated throughout the gland and in the pineal capsule. The number of IR nerve fibers in the capsule was high and from this location fibers were observed to penetrate into the gland proper via the pineal connective tissue septa, often following the blood vessels. From the connective tissue septa IR fibers intruded into the parenchyma between the pinealocytes. Many IR nerve fibers were observed in the pineal stalk and in the habenular as well as the posterior commissural areas. The number of NPY/CPON-IR nerve fibers in pineal glands from animals bilaterally ganglionectomized two weeks before sacrifice was low. The source of most of the extrasympathetic NPY/CPONergic nerve fibers is probably the brain from where they enter the pineal via the pineal stalk. However, an origin of some of the fibers from parasympathetic ganglia cannot be excluded due to the presence of a few IR fibers in the pineal capsule of ganglionectomized animals. It is concluded that the cat pineal is richly innervated with NPYergic nerve fibers mostly of sympathetic origin. The posttranslational processing of the NPY promolecule results in the presence of both NPY and CPON in intrapineal nerve fibers.     

Cytochemical studies on cytoplasmic granular elements in the hamster pineal gland

Summary The pineal gland of adult golden hamsters (Mesocricetus auratus) was studied by various cytochemical methods at the electron microscopic level: (1) the modified chromaffin reaction specific for 5-hydroxytryptamine (5-HT), (2) argentaffin reaction, (3) zinc-iodide-osmium (ZIO) mixture reaction and (4) acid phosphatase reaction. In the pinealocytes, the dense-cored vesicles (80–160 nm in diameter) show both chromaffinity and argentaffinity, while the population of dense bodies (150–400 nm in diameter) is reactive to ammoniacal silver solution and ZIO mixture but not to the modified chromaffin reaction. After incubation for demonstration of acid phosphatase activity, reaction products are localized in some, but not all, of the dense bodies, in some of the small vesicles in the Golgi region and in one or two inner Golgi saccules. In nerve fibers in the pineal gland, small granulated vesicles are also reactive to the modified chromaffin reaction and ZIO mixture. Based upon these cytochemical results the following conclusions have been reached: (1) dense cored vesicles in the pinealocytes and small granulated vesicles in the nerve fibers of the hamster pineal gland contain 5-HT, and (2) the population of dense bodies in the pinealocytes is heterogenous, some are lysosomes and the others are possibly the granules responsible for the secretion of pineal peptides.Supported in part by a grant from the National Science Council, Republic of ChinaDedicated to Professor Doctor Huoyao Wei on the occasion of his 70th birthday     

Glutamate immunoreactivity is enriched over pinealocytes of the gerbil pineal gland

Summary Mammalian pinealocytes have been shown to contain synaptic-like microvesicles with putative secretory functions. As a first step to elucidate the possibility that pinealocyte microvesicles store messenger molecules, such as neuroactive amino acids, we have studied the distributional pattern of glutamate immunoreactivity in the pineal gland of the Mongolian gerbil (Meriones unguiculatus) at both light- and electron-microscopic levels. In semithin sections of plastic-embedded pineals, strong glutamate immunoreactivity could be detected in pinealocytes throughout the pineal gland. The density of glutamate immunolabeling in pinealocytes varied among individual cells and was mostly paralled by the density of immunostaining for synaptophysin, a major integral membrane protein of synaptic and synaptic-like vesicles. Postembedding immunogold staining of ultrathin pineal sections revealed that gold particles were enriched over pinealocytes. In particular, a high degree of immunoreactivity was associated with accumulations of microvesicles that filled dilated process terminals of pinealocytes. A positive correlation between the number of gold particles and the packing density of microvesicles was found in three out of four process terminals analyzed. However, the level of glutamate immunoreactivity in pinealocyte process endings was lower than in presumed glutamatergic nerve terminals of the cerebellum and posterior pituitary. The present results provide some evidence for a microvesicular compartmentation of glutamate in pinealocytes. Our findings thus lend support to the hypothesis that glutamate serves as an intrapineal signal molecule of physiological relevance to the neuroendocrine functions of the gland.     

Transplantation of the rat pineal organ to the brain: pinealocyte differentiation and innervation

Summary The pineal organ of neonatal rats was transplanted to the frontal part of the cerebral cortex or the cerebral interhemispheric fissure of an isogenic adult rat to determine whether pineal differentiation and pinealopetal innervation are affected by aberrant neuronal influences. Transplants were fixed for immunohistochemistry at 1, 2 and 6 months after transplantation. When treated with an anti-serotonin antibody, cells in transplants from both locations showed intense immunoreactivity and a morphology comparable to intact pinealocytes, indicating that the transplanted pinealocytes had differentiated normally. Tyrosine hydroxylase immunohistochemistry revealed that new catecholamine fibers of central nervous origin extended only into the periphery and not into the core of transplants grafted within the cortex. However, numerous catecholamine fibers were found in transplants placed in the interhemispheric fissure. These fibers were often accompanied by blood vessels, suggesting that they derived from sympathetic ganglia. Serotonin fibers, which are densely distributed in the cerebral cortex, were seldom found to enter transplants from both locations. These observations indicate that pineal cells express their characteristic properties even when transferred to a foreign milieu and that they do not receive novel innervation from the central nerves that normally do not innervate the intact pineal body; the transplant thereby retains the property of selective pinealopetal innervation.