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.     

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

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

The pars intermedia of the mink,Mustela vison

Summary The pars intermedia of intact and experimental female minks has been studied by light, electron and fluorescence microscopy. The general structure of the mink intermediate lobe is described. Two main cell types are recognized. One, termed glandular cell, predominates in number and is characterized by the presence of electron-dense granules about 200 nm in diameter and numerous large vesicles up to 300 nm in diameter. The other, termed stellate cell, is devoid of cytoplasmic vesicles and granules and possesses microfilaments, junctional complexes and elongated processes inserted between the glandular cells. Different treatments (photostimulation and administration of hypertonic saline and metopirone) induced morphological reactions in the glandular cells. The significance of these changes and the possibility of a functional relation between the pars intermedia and ACTH secretion are discussed.Numerous nerve fibres and axon terminals containing electron-dense granules (60–120 nm) and electron-lucent vesicles (30–40 nm) are observed throughout the pars intermedia.With the histochemical fluorescence method of Falck-Hillarp a rich system of delicate fluorescent varicose fibres, sometimes provided with irregular swellings or droplets, is observed in the pars intermedia and also in the pars nervosa. Microspectrofluorometrically these fibres exhibit the spectral characteristics of catecholamines. All the cells of the pars intermedia and a large number of cells in the pars distalis show a yellowish weak fluorescence, which becomes much stronger after combined formaldehyde-ozone treatment. This cellular fluorophore shows the same microspectrofluorometric characteristics as does the fluorophores of tryptamine and of certain peptides with NH₂-terminal tryptophan.Supported by the Swedish Fur Breeders' Association and the Swedish Natural Science Research Council (grant No. 2124). Thanks are due to Miss W. Carlsson and Miss Y. Lilliemarck for their helpful technical assistance.Supported by the Harald and Greta Jeanssons Stiftelse and by the Ford Foundation. The skilful technical assistance of Mrs. Eva Svensson and Miss Annika Borgelin is greatfully acknowledged.     

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.     

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.     

The pineal gland of nocturnal mammals

Summary In the pineal gland of the pipistrelle bat two different populations of pinealocytes and glial cells were observed electron microscopically. The pinealocytes of populations I and II differ in their content of metabolically active cell organelles. In the pinealocytes of population I, granular vesicles originating from the Golgi apparatus were found in the perikaryon and especially in the endings of the pinealocyte processes. Granular vesicles appeared to be more numerous in hibernating nulliparous females. The pinealocytes of population II are characterized by the presence of small cytoplasmic vacuoles, probably originating from cisternae of the granular endoplasmic reticulum and containing flocculent material of moderate electron density. The classification of the pinealocytes belonging to population II is discussed.This collaboration was initiated with the aid of an SRC European short visit grant to P.A.R.The study was supported by the Foundation for Medical Research, the Netherlands (FUNGO, 13-35-33)     

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

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

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.     

Structure and innervation of the pineal gland of the rabbit,Oryctolagus cuniculus (L.)

Summary A light microscopic investigation of the rabbit pineal gland with the aid of silver-stained sections gave the following results. In the gland a medulla and a cortex can be distinguished, the medulla containing so-called light and dark pinealocytes, the cortex only light ones. Autonomic nerve fibres reach the pineal organ by two routes: (1) via the perivascular spaces of pineal blood vessels and (2) via two distinct nerve bundles, the nervi conarii. Bilateral superior cervical ganglionectomy revealed that these pinealo-petal nerve fibres are mainly orthosympathetic postganglionic. Intramural pineal neurones with synaptic-like structures on their cell bodies and dendrites point to the presence of a parasympathetic innervation next to the orthosympathetic one. Direct afferent or efferent neural connections with the brain appeared to be absent. Acknowledgements. The author wishes to thank Professor Dr. J. Ariëns Kappers for encouragement and help, Mr. H. K. Koerten for his technical assistance and Miss A. M. Feddema for typing the manuscipt.     

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 histochemical study of aldehyde fuchsin-positive material and "high-esterase cells" in the pineal gland of the Mongolian gerbil.

The pineal gland of the Mongolian gerbil consists of a superficial gland, stalk and deep pineal. The deep pineal differentiates postnatally. Histochemical studies of the superficial pineal gland indicate that it may be involved in the secretion of protein. Presumptive secretory material visualized by aldehyde fuchsin (AF) and chrome hematoxylin was observed along the course of blood vessels and among the pinealocytes. The distribution and texture of the AF-positive material was distinctive. It did not correspond to the pattern and texture of material stained with PAS, Sudan Black or acid orcein. Staining with AF was markedly reduced after incubation with trypsin, indicating that the AF-positive material is at least partially protein. The amount of stainable material increased with age. The AF-positive material was observed in what appeared to be interstitial or glial cells and processes, and in the processes of perivascular cells. Cells and fibrous processes with high non-specific esterase activity ("high-esterase cells") were observed among the pinealocytes and along the course of blood vessels. The distribution of the "high-esterase cells" and the morphology and texture of their esterase-containing processes were remarkably similar to the morphology and distribution of the material that stained with AF. It may be that the "high-esterase cells" contain AF-positive material. The "high-esterase cells" hydrolyzed both alpha-naphthyl acetate and alpha-naphthyl butyrate. The pinealocytes hydrolyzed only alpha-naphthyl acetate. The "high-esterase cells" appear to form a distinct class of cells within the superficial pineal gland. They are tentatively identified as a type of glial cell.     

Phenotypic expression of photoreceptor and endocrine cell properties by cultured pineal cells of the newborn rat

Pineal glands of newborn rats were dissociated and maintained under cell culture conditions. The phenotypic expression of both photoreceptor and endocrine cell properties was investigated using immunohistochemical techniques (specific antibodies against opsin or serotonin). After one week in culture, a number of small round cells appeared on top of a sheet of flat epithelium. Among those cells, opsin-like immunoreactive cells were observed. These cells showed a neuron-like morphology with neuritic processes and often formed rosettes. Immunoreactivity was found on the plasma membrane of both the soma and cell processes. Serotonin-like immunoreactive cells were also differentiated in culture with two different morphological types of cells being found. One type resembled cultured serotonin-containing amacrine cells of the retina, and the other type had a flat, polygonal shape similar to that of pinealocytes. Both types of immunoreactive cells possessed fine neuritic processes. These results indicated that cell culture of rat pineal gland cells allowed expression of some properties, such as opsin synthesis and neuron-like morphology with long neuritic processes, that were not expressed in the intact rat pineal gland.     

Distribution of hydroxyindole-O-methyltransferase mRNA in the rat brain: an in situ hybridisation study

Hydroxyindole-O-methyltransferase (HIOMT) is the enzyme involved in the last step of the melatonin synthesis pathway. Recently, a cDNA encoding HIOMT has been isolated from a rat pineal gland library. Using this cDNA, we developed a highly sensitive in situ hybridisation protocol to investigate the distribution of HIOMT mRNA in both the rat brain and dissociated pinealocytes maintained in primary cell culture. In the rat brain, HIOMT mRNA was only detected in the three parts of the pineal complex: the superficial pineal, the stalk and the deep pineal. No extra-pineal hybridisation labelling was observed. These results strongly suggest that melatonin synthesis also occurs in the deep part and the stalk of the pineal gland. HIOMT mRNA was markedly expressed in cultured pinealocytes. No particular subcellular area was observed to express HIOMT mRNA specifically, as the labelling was homogeneously distributed in the cytosol and in the axon-like processes. In conclusion, the use of in situ and in vitro hybridisation with a pineal riboprobe has detected notable HIOMT expression restricted to pinealocytes. Received: 26 June 1997 / Accepted: 15 September 1997     

Observations on the fine structure,enzyme histochemistry,and innervation of parathyroid gland and ultimobranchial body of Chthonerpeton indistinctum (Gymnophiona,Amphibia)

Summary Fine structural and enzyme histochemical observations on ultimobranchial body and parathyroid gland of the caecilian Chthonerpeton are presented. The cell clusters and follicles of the ultimobranchial body consist mainly of granulated cells which are termed C-cells and obviously belong to the APUD cell series. In the larger follicles additional possibly exhausted degranulated cells and replacement cells occur. A rich supply of nerve fibres has been found in this gland. Frequently nerve terminals were observed to come into synaptic contact with the C-cells. Two categories of nerve fibres occur: a) fibres containing large polymorphic electron dense granules (probably purinergic fibres), b) fibres containing small electron transparent vesicles and a few electron dense granules (probably cholinergic fibres). The parathyroid gland consists of elongated cells (one cell type) poor in organelles and often containing fields of glycogen and lipid droplets. The cells are further characterized by fair amounts of lysosomal enzymes; they are interconnected by maculae adhaerentes and occludentes. No nerves and blood vessels have been found in the parathyroid gland of Chthonerpeton. This study has been supported by the Deutsche Forschungsgemeinschaft We 380/5.     

蒙古黄鼠(Citellus dauricus)松果腺的超微结构观察

蒙古黄鼠松果腺主要由低电子密度的松果腺细胞和少量的胶质细胞、含色素细胞、神经突起及血管等组成。松果腺细胞内含有大量的线粒体、溶酶体、微丝、高尔基器、游离核糖体及中等量的光面和粗面内质网。纤毛、中心粒、突触带和致密芯小泡很少。松果腺细胞之间及胶质细胞之间存在电突触。最新被观察到的是大约有5%松果腺细胞内的线粒体产生“融合”现象,形成类似电突触的结构。神经突起可形成轴—轴突触,轴—树突触,并与松果腺细胞形成突触。     

蒙古黄鼠（Citellus dauricus）松果腺的超微结构观察

The distal part of pineal gland of the Mongolian ground squirrel was ultrastructurally studied. The gland was composed of low electron-dense parenchymal cells, among which glial cells, pigment cells, blood vessels and neural elements were occasionally interspersed. The pinealocytes contained numerous mitochondria, lysosomes, microtubules, microfilaments, Golgi apparatus and free ribosomes, as well as less prominent profiles of rough- and smooth-surfaced endoplasmic reticula and some cilia, centrioles, synaptic ribbons and few subsurface cisterns. Some pinealocytes were vacuolated. The content of the vacuoles released into the extracellular space by exocytosis could be observed. The gap junctions between pinealocytes were also observed. Of particular interest was that many mitochondria "fused together" and formed gap junction-like structure in about five percent pinealocytes. The pigment cell has a amorphous nucleus which contains many aggregated chromatin, its cell membrane has a few microvilli projecting into a central lumen, these features may indicated that this kind of cell differs either from the pinealocyte or astrocyte. There are axo-axonic synapses or axo-dendritic synapses between neuron processes or between neuron processes and pinealocytes.     

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.     

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.     

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

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

The pineal gland of the horse. Morphological and histochemical results. (With notes on the donkey and mule pineal)

The horse pineal gland has been investigated by morphological and histochemical methods. Particular care has been given to the cellular types, to the eventual presence of neurosecretory activity and to the nature of the pigments. Even in the horse pineal, it is possible to distinguish two populations of pinealocytes, morphologically but not histochemically distinct. A great number of pinealocytes are positive for the Masson- Hamperl reaction, and for Gomori- Bargmann 's chromic haematoxylin-phloxine and Gomori's paraldehyde-fuchsin. Along the connective septa, many brown- blackish pigmented cells were present; their pigment was positive for the Lillie and the Masson-Fontana reactions for the determination of melanin pigment. Another type of pigmented cells, carrying a brown yellowish pigment of lipofuscin nature was present, particularly in older animals, along the connective septa.