Interstitial and parenchymal cells in the pineal gland of the golden hamster

Summary A combined thin-section/freeze-fracture study was performed on the superficial pineal gland of the golden hamster, comparing the parenchymal and interstitial cells of this animal with those previously investigated in rats. In contrast to rats, no gap junctions and gap/tight junction combinations could be found between pineal parenchymal cells of the hamster. Furthermore, the interstitial cells of the hamster pineal gland were found to have large flat cytoplasmic processes, which abut over large areas equipped with tight junctions. In thin sections, profiles of interstitial cell processes were seen to surround groups of pinealocytes. Interstitial cells and their sheet-like, tight junction-sealed processes thus appear to delimit lobule-like compartments of the hamster pineal gland. Because the classification of the interstitial cells is uncertain, the expression of several markers characteristic of mature and immature astrocytes and astrocyte subpopulations has been investigated by indirect immunohistology. Many of the non-neuronal elements in the pineal gland are vimentin-positive glial cells, subpopulations of which express glial fibrillary acidic protein (GFA) and C1 antigen. The astroglial character of these cells is supported by the lack of expression of markers for neuronal, meningeal and endothelial cells. M1 antigen-positive cells have not been detected.Supported by a grant from Deutsche Forschungsgemeinschaft (Scha 185/9-2)     

Characterization of rodent pineal astrocytes by immunofluorescence microscopy using a monoclonal antibody (J1-31)

Summary In previous studies pineal astrocytes have been characterized immunohistochemically mainly by use of antisera to glial fibrillary acidic protein. Because of the recent demonstration of this protein in non-astrocytic cells the question of its specificity as an astrocytic marker has been raised. A possible alternative tool for characterizing pineal astrocytes is the J1-31 monoclonal antibody, which is directed against a 30 000 dalton astrocytic protein clearly distinguishable from glial fibrillary acidic protein. Immunofluorescence microscopy of this antibody in the pineal gland of rat and guinea-pig revealed a staining pattern similar to that obtained by glial acidic fibrillary protein antisera. In the rat, J1-31-immunoreactive cells and processes were concentrated in the transitional region between the superficial pineal gland and pineal stalk. Fibrillar J1-31-immunoreactive structures were seen in the most proximal part of the guinea-pig pineal gland. The J1-31 monoclonal antibody therefore appears to be a useful tool for the demonstration of pineal astrocytes; it avoids the specificity problems of glial fibrillary acidic protein immunohistochemistry.Supported by the Deutsche Forschungsgemeinschaft, grant Schr 283/2-1, NSERC (A 5021) and MSI Foundation     

Ultrastructural and immunocytochemical characterization of interstitial cells in pre- and postnatal developing sheep pineal gland.

Pineal gland interstitial cells from 32 sheep embryos (from day 54 of gestation until birth) and 18 sheep (from 1 month to >2 years) were analysed using ultrastructural and immunohistochemical techniques. From day 98 of gestation and throughout postnatal development, a second cell type was observed in addition to pinealocytes; these cells displayed uniform ultrastructural features similar to those of CNS astrocytes. Ultrastructural homogeneity was not matched by the results of histochemical and immunohistochemical analysis. Expression of phosphotungstic acid hematoxylin, glial fibrillary acidic protein and vimentin indicates that the second cell population in the developing ovine pineal gland is, in fact, a combination of glial-astrocyte cells at varying stages of maturity. Pineal interstitial cells started to show signs of functional activity evident in vascular tropism; such activity, evident from around day 98 of gestation, appeared to relate to the exchange of substances between the pineal parenchyma and blood vessels and, though it continued throughout postnatal development, was most evident in animals slaughtered between 9 months and 2 years of age (group II). Morphologically, functional activity in interstitial cells in this age-group was apparent in: 1, formation of specific contact sites between interstitial cells and nerve fibres in the perivascular space; and 2, the presence of numerous gap junctions between the bulbous endings of cytoplasmic processes.     

Determination of pineal melatonin by precolumn derivatization reversed-phase high-performance liquid chromatography and its application to the study of circadian rhythm in rats and mice

Determination of minute amounts of endogenous melatonin in rat and mouse pineal gland was performed using an RP-HPLC system. Melatonin was separated following precolumn derivatization and determined with a fluorescence detector at the emission wavelength of 380 nm with the excitation at 245 nm. The calibration curve of melatonin constructed by adding known amounts of melatonin to the homogenates of mouse pineal gland was linear over the range of 1-500 fmol (injection amount/20 microl). The detection limit of added melatonin was 1 fmol (S/N = 5). Repeatability and day-to-day precision for the melatonin spiked sample of mouse pineal gland was 4.0 and 3.8% (RSD), respectively. Using the present method, circadian changes of melatonin content in rat (Wistar) and mouse (C3H) pineal gland were determined. In addition, a minute amount of melatonin in ddY mouse pineal gland was determined, because pineal melatonin of many inbred mouse strains has been reported to be lower than the detection limit.     

Immuno-electron-microscopic localization of enkephalin in the secretory granules of C cells in the chicken ultimobranchial glands

Interstitial cells in the pineal gland of the rat were characterized immunocytochemically using the monoclonal antibodies MRC OX-42 and ED1 for macrophages/microglia, and MRC OX-6, which recognizes major histocompatibility complex (MHC) class II antigen. A polyclonal antibody against GFAP was used to identify astrocytes. Cells immunopositive for OX-42 and/or ED1 were distributed throughout the gland; they extended processes primarily along the perivascular spaces and occasionally within the parenchyma of the gland. Ultrastructurally, these OX-42-positive cells were characterized by a nucleus with sparse heterochromatin and cytoplasmic vacuoles/lysosomes. Cells expressing MHC class II antigen had a distribution and morphology similar to OX-42-immunopositive cells, suggesting that pineal macrophages/microglia play a role as antigen-presenting cells. GFAP-positive astrocytes were concentrated at the proximal end of the pineal where the pineal stalk enters the gland. The occurrence of antigenpresenting cells in the circumventricular neuroendocrine gland has important functional implications as these cells may be mediators of neuroimmunomodulatory mechanisms, and involved in certain disease states such as autoimmune pinealitis.     

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.     

S-antigen and glial fibrillary acidic protein immunoreactivity in the in situ pineal gland of hamster and gerbil and in pineal grafts: developmental expression of pinealocyte and glial markers.

Postnatal development of S-Ag and GFAP immunoreactivity in the in situ pineal glands of golden hamsters and gerbils was examined using the avidin-biotin-peroxidase immunohistochemical technique. S-Ag was present in the gerbil pineal gland on the first postnatal day (P1), whereas it did not appear in the hamster pineal until P6. GFAP-immunoreactive astrocytes were first observed in the hamster pineal gland on P7 and in the gerbil pineal gland on P10. The number of S-Ag-immunoreactive pinealocytes and GFAP-immunoreactive astrocytes in the pineal glands of hamsters and gerbils increased with increasing age from P7 to 3 weeks. By 4 weeks, strong S-Ag and GFAP immunoreactivity was observed in both hamster and gerbil pineal glands. GFAP-immunoreactive stellate astrocytes were distributed evenly throughout the gerbil superficial pineal gland, but they were more often located in the peripheral region of the hamster superficial pineal. For the pineal grafts, pineal glands from neonatal (3-5 day old) hamsters were transplanted into the third cerebral ventricle (infundibular recess or posterior third ventricle) or beneath the renal capsule of adult male hamsters. S-Ag immunoreactivity appeared in the pineal grafts within 1 week following transplantation. By 4 weeks the pineal grafts showed strong S-Ag immunoreactivity which was maintained until at least 12 weeks after transplantation. The time course of glial cell maturation in the cerebroventricular pineal grafts is generally parallel to the hamster pineal gland in situ before 4 weeks. By 12 weeks, however, more astrocytes differentiated and developed GFAP-immunoreactivity in the pineal grafts than in the in situ pineals. These studies have described the postnatal development of S-Ag and GFAP immunoreactivity in in situ pineal glands and in neonatal pineal grafts.     

Species differences in reactivity of mouse and rat astrocytes in vitro

Reactive astrogliosis constitutes a major obstacle to neuronal regeneration and is characterized by rearrangement and upregulation of expression of cytoskeletal proteins, increased proliferation and hypertrophy. Many approaches have been attempted to mimic astrogliosis by inducing reactive astrocytes in vitro. Such research is usually performed using astrocytes derived from Mus musculus or Rattus norvegicus, and results compared between species on the assumption that these cells behave equivalently. Therefore, we compared reactivity between mouse and rat astrocytes in scratch wound assays to gain further insight into how comparable these cell culture models are. Proliferation and migration, as well as expression of the cytoskeletal proteins glial fibrillary acidic protein (GFAP) and vimentin, were compared by immunocytochemistry and immunoblot. Further, we investigated migration of proliferating cells by 5-ethynyl-2'-deoxyuridine staining. Substantial differences in GFAP expression and proliferation between astrocytes of the two species were found: rat astrocytes showed different cytoskeletal morphology, expressed significantly more GFAP and vimentin of different molecular size and were more proliferative than comparable mouse astrocytes. Our results suggest that rat and mouse astrocytes may respond differently to various reactivity-triggering stimuli, which needs to be considered when general conclusions are drawn regarding effects of factors regulating astrocyte reactivity.     

The Perivascular Phagocyte of the Mouse Pineal Gland: an Antigen‐Presenting Cell

The perivascular space of the rat pineal gland is known to contain phagocytic cells that are immunoreactive for leukocyte antigens, and thus they appear to belong to the macrophage/microglial cell line. These cells also contain MHC class II proteins. We investigated this cell type in the pineal gland of mice. Actively phagocytosing cells with a prominent lysosomal system were found in the pericapillary spaces of the mouse pineal gland following intravenous injection of horseradish peroxidase. The cells also exhibited strong acid phosphatase activity. Perivascular cells were immunopositive for MHC class II protein and for CD68, a marker of monocytes/phagocytes. This study verifies that perivascular phagocytes with antigen‐presenting properties are present in the mouse pineal gland.     

The perivascular phagocyte of the mouse pineal gland: an antigen-presenting cell

The perivascular space of the rat pineal gland is known to contain phagocytic cells that are immunoreactive for leukocyte antigens, and thus they appear to belong to the macrophage/microglial cell line. These cells also contain MHC class II proteins. We investigated this cell type in the pineal gland of mice. Actively phagocytosing cells with a prominent lysosomal system were found in the pericapillary spaces of the mouse pineal gland following intravenous injection of horseradish peroxidase. The cells also exhibited strong acid phosphatase activity. Perivascular cells were immunopositive for MHC class II protein and for CD68, a marker of monocytes/phagocytes. This study verifies that perivascular phagocytes with antigen-presenting properties are present in the mouse pineal gland.     

Postnatal development of female sheep pineal gland under natural inhibitory photoperiods: an immunocytochemical and physiological (melatonin concentration) study

The purpose of this study was to determine structural and immunocytochemical changes taking place during the day and at night in developing sheep pineal gland under natural non-stimulatory photoperiods (summer solstice). Additionally, the diurnal cycle of plasma melatonin levels was charted and differences between diurnal and nocturnal pineal melatonin concentrations were analyzed. 36 ewes of three different ages were examined: infants (1-6 months old), pubertal and early fertile age (9-24 months old) and adults (36-60 months old). Plasma and pineal gland melatonin levels were higher in pubertal sheep than in infants or adults. Pubertal sheep pineal glands were also heavier, contained a larger number of pinealocytes and interstitial cells and displayed more evident innervation and vascularisation than infants or adults. There was no difference in the number of pinealocytes and interstitial cells between animals killed during daylight or at night. Gland weight, pinealocyte nuclear profile areas and plasma melatonin concentrations were all significantly higher at night than during the day.     

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.     

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.     

Differential Expression of mRNA and Protein Encoding Retinal and Pineal S-Antigen During the Light/Dark Cycle

S-Antigen is a soluble cell protein unique to the retina and pineal gland. In the former, it is a well-characterized molecule that participates in light-induced signal transduction in photoreceptor cells. In the latter, the functional role is presently not known. The expression of S-antigen and its mRNA was examined in the rat retina and pineal gland throughout the diurnal cycle and with light interruption of the dark cycle. A cDNA for rat S-antigen was isolated from a pineal gland library to examine the mRNAs. A 1.7-kb mRNA for S-antigen was observed in both the pineal gland and the retina. Retinal S-antigen mRNA was expressed throughout the diurnal cycle and increased with light interruption of the dark cycle. In contrast, pineal gland S-antigen mRNA levels were detectable only during the dark and were absent preceding and during light. The phenotypic expression of immunoreactive S-antigen, identified with two S-antigen monoclonal antibodies (MAbs), MAb A9C6 and MAb C10C10, was analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel (PAGE) and isoelectric focusing (IEF) electrophoresis. Immunoblot analysis of gels after SDS-PAGE revealed a single 46-kDa protein in retina. In contrast, two bands of approximately 43 and 46 kDa were identified in the pineal gland. Immunoblots of the retinal extracts separated by IEF electrophoresis revealed five S-antigen isomers, which vary quantitatively throughout the diurnal cycle and when light interrupted the dark cycle. Immunoblots of the pineal gland samples separated by IEF electrophoresis indicated that the pineal gland possesses four pineal gland-specific forms of S-antigen in addition to the five forms present in the retina. The differences observed in the mRNA and protein analyses suggest tissue-specific structural components for S-antigen in the retina and pineal gland that are not regulated in the same manner.     

Astrocytes produce and secrete FGF-1, which promotes the production of apoE-HDL in a manner of autocrine action

The astrocytes prepared by 1 week secondary culture after 1 month primary culture of rat brain cells (M/W cells) synthesized and secreted apolipoprotein E (apoE) and cholesterol more than the astrocytes prepared by conventional 1 week primary and 1 week secondary culture (W/W cells) (Ueno, S., J. Ito, Y. Nagayasu, T. Furukawa, and S. Yokoyama. 2002. An acidic fibroblast growth factor-like factor secreted into the brain cell culture medium upregulates apoE synthesis, HDL secretion and cholesterol metabolism in rat astrocytes. Biochim. Biophys. Acta. 1589: 261-272). M/W cells also highly expressed fibroblast growth factor-1 (FGF-1) mRNA. FGF-1 was identified in the cell lysate of both cell types, but M/W cells released more of it into the medium. Immunostaining of FGF-1 and apoE revealed that both localized in the cells that produce glial fibrillary acidic protein. The conditioned media of M/W cells and FGF-1 stimulated W/W cells to release apoE and cholesterol to generate more HDL. Pretreatment with a goat anti-FGF-1 antibody or heparin depleted the stimulatory activity of M/W cell-conditioned medium. The presence of the anti-FGF-1 antibody in the medium suppressed apoE secretion by M/W cells. Differential inhibition of signaling pathways suggested that FGF-1 stimulates apoE synthesis via the phosphoinositide 3-OH kinase for PI3K/Akt pathway. Thus, astrocytes release FGF-1, which promotes apoE-HDL production by an autocrine mechanism. These results are consistent with our in vivo observation that astrocytes produce FGF-1 before the increase of apoE in the postinjury lesion of the mouse brain (Tada, T., J. Ito, M. Asai, and S. Yokoyama. 2004. Fibroblast growth factor 1 is produced prior to apolipoprotein E in the astrocytes after cryo-injury of mouse brain. Neurochem. Int. 45: 23-30).     

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

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