Showing papers on "Pinealocyte published in 1993"

Distribution of melatonin in mammalian tissues: The relative importance of nuclear versus cytosolic localization

Abstract: Besides its presence in the pineal gland, melatonin has been found in a variety of other tissues as well. The indoleamine also has been identified in invertebrates including an unicellular organism where it exhibits a diurnal rhythm. Although melatonin is mainly known for its effects on seasonal reproduction and endocrine physiology, there is evidence showing that this ubiquitously acting hormone is also a potent free radical scavenger, thereby providing protection from oxidative attack to DNA and other biomolecules. Through the years, melatonin was thought to be exclusively cytosolic. However, careful examination of some of these pioneering reports revealed a nuclear localization of melatonin in different tissues including the retina and Harderian glands. Using a very sensitive immunocytochemical method, we have also found that melatonin is located in the nucleus of many cells where it may bind to nuclear components. The use of cell fractionation studies followed by radioimmunoassay confirmed these results. The administration of exogenous melatonin resulted in a marked increase in the nuclear melatonin content without a concomitant change in the cytosolic fraction. In addition to its ability to scavenge free radicals, its location in the nucleus suggests possible genomic actions.

The contribution of extrapineal sites of melatonin synthesis to circulating melatonin levels in higher vertebrates

Abstract: While the production of melatonin in higher vertebrates occurs in other organs and tissues besides the pineal, the contribution of extrapineal sites of melatonin synthesis such as the retina, the Harderian glands and the gut to circulating melatonin levels is still a matter of debate. The amount of melatonin found in the gastrointestinal tract is much higher than in any other organ including the pineal and the gut appears to make a significant contribution to circulating melatonin at least under certain conditions. The gut has been identified to be the major source of the elevated plasma concentrations of melatonin seen after tryptophan administration and of the changes of circulating melatonin level induced by the feeding regimen. Whereas the circadian and circannual fluctuations of the concentration of melatonin in the blood seem to be triggered by changes of the photoenvironment and its effect of pineal melatonin formation, basal daytime melatonin levels and the extent of their elevation at nighttime appear to be additionally controlled by nutritional factors, such as the amount and the composition of ingested food and therefore availability of tryptophan as a rate-limiting precursor of melatonin formation by the enterochromaffin cells of the gastrointestinal tract.

Interactions of the pineal hormone melatonin with oxygen-centered free radicals: a brief review.

Abstract: Melatonin,N-acetyl-5-methoxytryptamine, is a hormonal product of the pineal gland. Its synthesis is higher at night than during the day in all vertebrates including man. Once melatonin is produced in the pineal gland it is quickly released into the vascular system. The rapid release of melatonin is generally believed to relate to its high lipophilicity which allows it to readily pass through the membrane of the pinealocytes and the endothelial cells which line the capillaries. The result of the nocturnal synthesis and secretion of melatonin is high blood levels at night. Also because of its highly lipophilic nature, melatonin from the blood readily escapes into every other bodily fluid and all cells in the body. Until recently it was generally thought that melatonin's action in the organism depended on its exclusive interaction with specific receptors on cells located in discrete locations. Certainly, the interactions of melatonin with these membrane-bound receptors are believed to mediate the endocrine and circadian rhythm effects of melatonin. It was recently discovered, however, that melatonin's primary action may not depend on the previously described membrane receptors. We have found that melatonin is a very potent hydroxyl radical scavenger; free radicals and the hydroxyl radical in particular, because of its very high reactivity, can be extremely damaging to macromolecules in cells. Compared to glutathione and mannitol, two well known free radical scavengers, melatonin is a more powerful scavenger and affords protection of molecules, especially DNA, from oxidative damage. Melatonin's extremely high diffusibility is important for its scavenging action because this feature allows it to enter all cells and every subcellular compartment. Whereas the free radical quenching activity of melatonin does not require a receptor, we also have evidence that it may be bound in the nucleus thereby providing on-site protection to DNA. Besides scavenging the highly toxic hydroxyl radical, melatonin also stimulates glutathione peroxidase activity which metabolizes the precursor of the hydroxyl radical, hydrogen peroxide, to water. Thus, melatonin has at least two means to protect the cell from oxidative damage, i.e., it breaks down hydrogen peroxide to harmless water and, in the event any hydroxyl radicals are formed, melatonin scavenges them. Melatonin may be the premier molecule to protect the organism from oxidative damage.

Melatonin Receptors in Peripheral Tissues: A New Area of Melatonin Research

Abstract: Melatonin is synthesized and secreted by pineal gland, retina, harderian gland, lacrimal gland, and gut. It is a lipid-soluble molecule with ubiquitous distribution and multifarious functions. Thus, along with the better established sites of melatonin action, such as brain, retina, and pituitary, direct melatonin actions on other peripheral tissues should also be considered. The presence of melatonin receptors in peripheral tissues was rightfully hypothesized. Earlier studies on melatonin receptors had limited success. The advent of 2-[125I]iodomelatonin, a labelled melatonin agonist with a specific activity as high as 2,200 Ci/mmol, has allowed the studies of melatonin receptors with picomolar affinity and femtomolar density. Putative melatonin receptors demonstrated by autoradiography and/or radioreceptor assay in gut, kidney, lung, heart, vas deferens, and blood vessels are discussed.

Synaptophysin – a common constituent of presumptive secretory microvesicles in the mammalian pinealocyte: A study of rat and gerbil pineal glands

Abstract: Recent studies have established that pinealocytes of the mammalian pineal gland contain marker molecules of neuroendocrine cells or paraneurons like the synaptic vesicle-associated protein synaptophysin (p38). The objective of this study was to identify the subcellular synaptophysin-positive compartment and to characterize in detail the intracellular distribution of this protein in rat and gerbil pinealocytes. An analysis of serial semithin sections of plastic-embedded pineals immunostained for synaptophysin, including computer-assisted optical density measurements of synaptophysin immunoreactivities, demonstrated unequivocally that synaptophysin was highly concentrated in dilated process terminals of the pinealocytes. More than 75% of these process terminals were found to border or lie within the pericapillary space. At the ultrastructural level, they contained accumulations of small clear vesicles of variable size that turned out to be the site of synaptophysin immunoreactivity when immunogold staining was performed. In addition, microvesicles surrounding synaptic ribbons were also immunolabeled. Hence, the pinealocyte is the first neuroendocrine cell type that has now been shown to concentrate synaptophysin-positive microvesicles in perivascular process endings. This observation lends strong support to the hypothesis that small clear vesicles in neuroendocrine cells in general, and in pinealocytes in particular, serve secretory functions. The quantitative analysis of completely sectioned process endings revealed that the microvesicles outnumber by far the amount of dense core vesicles and therefore cannot arise by endocytosis of dense core vesicle membranes. Thus, small synaptic-like vesicles probably constitute an independent secretory pathway of the paraneuronal pinealocytes. In the present study, we could also establish the absence of immunoreactivity for synapsin I (belonging to a family of neuron-specific nerve terminal phosphoproteins) from pinealocytes. Synapsin I immunoreactivity was only detectable in intrapineal nerve terminals and varicosities. Taken together, the immunostaining patterns of the pineal gland obtained with antibodies directed against synaptic vesicle-associated proteins render the mammalian pinealocyte a very special type of neuroendocrine cell or paraneuron rather than a "classic" neuron.

Microdialysis of melatonin in the rat pineal-gland - methodology and pharmacological applications

Abstract: The present study describes the development of a new technique to measure melatonin contents in the pineal gland of freely moving rats, by means of on-line microdialysis. The transcerebral cannula was modified, and a sensitive assay of melatonin, using HPLC with fluorimetric detection, was set up. With this system it is possible to monitor the melatonin levels on-line in the pineal gland during day- and nighttime. The nightly increase in melatonin release was recorded. Tetrodotoxin had an inhibitory effect on nighttime levels, whereas even high concentrations did not alter the daytime level. From this we conclude that neuronal activity is necessary to synthesize melatonin and that during daytime no net neuronal activity is present. Melatonin levels could be greatly enhanced by systemic administration of the beta-agonist isoprenaline (ISO). Also, local infusion of ISO or 8-bromoadenosine 3',5'-cyclic monophosphate, an analogue of the second messenger cyclic AMP, resulted in increased melatonin levels, demonstrating the presence of beta-adrenergic receptors, coupled to a cyclic AMP-based second messenger system, on the pineal gland. Injection of phenylephrine had no effect on daytime levels. Only when administered during ISO-induced stimulation of melatonin release did it enhance this stimulated release. This proved the regulatory role of alpha1-receptors on pinealocytes. The method presented is of special interest for investigating the innervation of the pineal gland and the biochemical processes that regulate the biosynthesis of melatonin. Also, for studies on the diurnal rhythms of melatonin release and factors that influence these rhythms in freely moving animals, this model will be of great value.

Adrenergic Stimulation of Cyclic GMP Formation Requires NO‐Dependent Activation of Cytosolic Guanylate Cyclase in Rat Pinealocytes

Abstract: Cyclic GMP (cGMP) formation in rat pinealocytes is regulated through a synergistic dual receptor mechanism involving beta- and alpha 1-adrenergic receptors. The effects of NG-monomethyl-L-arginine (NMMA), which inhibits nitric oxide (NO) synthase and NO-mediated activation of cytosolic guanylate cyclase, and methylene blue (MB), which inhibits cytosolic guanylate cyclase, were investigated in an attempt to understand the role of NO in adrenergic cGMP formation. Both NMMA and MB inhibited beta-adrenergic stimulation of cGMP formation as well as alpha 1-adrenergic potentiation of beta-adrenergic stimulation of cGMP formation, whereas they had no effect in unstimulated pinealocytes. The inhibitory action of NMMA was antagonized by addition of L-arginine. On the basis of these findings it can be concluded that the adrenergic stimulation of cGMP formation involves NO synthesis followed by activation of cytosolic guanylate cyclase.

Structures and molecules involved in generation and regulation of biological rhythms in vertebrates and invertebrates.

Abstract: Melatonin from the retina and the pineal gland functions in neuroendocrine hierarchies. Photoreceptors — eyes and extraretinal — detect light. Oscillators — pineal and suprachiasmatic nuclei — act as pacemakers. Driven neuroendocrine rhythms carry temporal hormone signals throughout the body. Light controls melatonin: light sets the phase of the melatonin rhythm and determines the duration of melatonin synthesis. By these means, circadian rhythms (e.g. in locomotor activity and body temperature) and seasonal rhythms (e.g. in reproduction) are controlled.

Vasoactive intestinal peptide stimulation of cyclic guanosine monophosphate formation: further evidence for a role of nitric oxide synthase and cytosolic guanylate cyclase in rat pinealocytes.

Abstract: In the rat pineal gland vasoactive intestinal peptide (VIP) and beta-adrenergic agonists stimulate cyclic guanosine monophosphate (cGMP) formation and their action is amplified by alpha 1-adrenergic agonists. Since beta-adrenergic stimulation of cGMP is suggested to involve activation of nitric oxide (NO) synthase and NO-mediated activation of cytosolic guanylate cyclase (GC), we investigated the effects of the NO synthase inhibitor N-monomethyl-L-arginine (L-NMMA) and of the cytosolic GC inhibitor methylene blue (MB) on VIP receptor-stimulated cGMP formation. Both L-NMMA and MB depressed VIP-induced cGMP formation as well as alpha 1-adrenergic potentiation of VIP-stimulated cGMP formation to the level of unstimulated pinealocytes. Further, L-arginine (L-arg) antagonized the effect of L-NMMA. However, L-arg did not antagonize the effect of MB, indicating that activation of NO synthase does not appear to compensate inhibition of cytosolic GC. On the basis of these findings it is concluded that VIP-stimulat...

The Mammalian Pineal Gland as an End Organ of the Visual System

Abstract: Visible light is the major environmental factor controlling the circadian production of melatonin in the mammalian pineal gland. Regardless of the activity pattern a species displays, i.e. diurnal, nocturnal or crepuscular, high melatonin production is always associated with the dark phase of the light: dark cycle. Light has two effects on the circadian melatonin rhythm; it precisely entrains the rhythm to 24 hours and light exposure during the night rapidly depresses high nighttime melatonin levels. The intensity of light required to inhibit nocturnal melatonin production varies widely among species with melatonin synthesis in the pineal gland of nocturnal animals having a much greater sensitivity (lower threshold) to light than that of diurnally active animals. While optic enucleation prevents all actions of light on the 24 hour melatonin cycle, destruction of the majority, if not all, the retinal photoreceptors does not alter the ability of light to synchronize the melatonin rhythm and only slightly increases the threshold of the pineal gland to acute light exposure. Beside luminance or intensity, the specific wavelength of light is important in determining its efficacy as an inhibitor of pineal melatonin synthesis. In general, blue-green wavelengths seem to be most inhibitory to the melatonin forming ability of the pineal. Red light (>600 nm) exposure, however, also suppresses pineal and serum melatonin levels. The ability of red light to achieve this effect is retained in rats with total or near total destruction of the retinal photoreceptors. To date there is no adequate explanation as to the retinal mechanisms involved in mediating the inhibitory effect of light on the pineal gland. It is possible, however, that the classical photoreceptors, i.e. the rods and cones, may not be involved in this process.

Developmental appearance of pineal adrenergic-->guanosine 3',5'-monophosphate response is determined by a process down-stream from elevation of intracellular Ca2+: possible involvement of a diffusible factor.

Abstract: Adrenergic stimulation of the adult pineal gland increases cAMP and cGMP production by over 100-fold. beta-Adrenergic stimulation results in Gs alpha-mediated cyclase activation; alpha 1-adrenergic activation potentiates the beta-adrenergic effects through mechanisms mediated by the intracellular Ca2+ concentration ([Ca2+]i) and Ca(2+)-phospholipid-dependent protein kinase. Development analysis of these responses has indicated that the adrenergic stimulation of cAMP is present several days after birth, but the cGMP response develops only after the second week of life. In the study presented here, the adrenergic-->cGMP response was analyzed in pineal glands from 10- and 25-day-old rats, with the intention of determining the basis of the developmental appearance of this response. Organ culture and tissue homogenate studies indicated that guanylate cyclase activity, cGMP phosphodiesterase activity, and adrenergic elevation of phospholipase-A2 were similar in pineal glands from 10- and 25-day-old rats. Norepinephrine stimulated an increase in [Ca2+]i in dispersed pinealocytes from 10-day-old rats, as has been previously demonstrated in adult pinealocytes. In contrast, several treatments that elevate [Ca2+]i had no effect on cGMP accumulation in forskolin-treated or beta-adrenergically activated glands from 10-day-old rats, but were fully effective in similarly treated glands from 25-day-old rats. However, glands from 10-day-old animals showed a 33-fold accumulation of cGMP when they were cultured together with glands from 25-day-old rats. These studies indicate that whereas many elements in the system that mediate adrenergic regulation of pineal cGMP are fully developed at 10 days of age, the developmental appearance of the cGMP response is triggered by the development of a process down-stream of the alpha 1-adrenergic stimulation of [Ca2+]i, and this process may involve a diffusible factor.

Rod-opsin immunoreaction in the pineal organ of the pigmented mouse does not indicate the presence of a functional photopigment

Abstract: The aim of the present study was to characterize the rod-opsin immunoreaction in the mammalian pineal organ. Pigmented mice (strain C57BL) were selected as the animal model. Immunocytochemical investigations involving the use of highly specific polyclonal and monoclonal antibodies against bovine rod-opsin (the apoprotein of the photopigment rhodopsin) showed that approximately 25% of all pinealocytes were rod-opsin immunoreactive. Immunoblotting techniques revealed three protein bands of approximately 40, 75, and 110 kDa; these were detected by the monoclonal antibody and the polyclonal antiserum in retinal and pineal extracts. These protein bands presumably represented the monomeric, dimeric and trimeric forms of rod-opsin. The amount of rod-opsin in retina and pineal organ was quantified by means of an enzyme-linked immunosorbent assay. This yielded 570±30 pmoles rod-opsin per eye and 0.3±0.05 pmoles rod-opsin per pineal organ. High pressure liquid chromatography analysis of whole eye extracts demonstrated the chromophoric group of the photopigment rhodopsin, 11-cis retinal, and its isomer, all-trans-retinal. A shift from 11-cis retinal to all-trans-retinal was found upon light adaptation. No retinals were detected in the pineal organ. Autoradiographic investigations showed that 3H-retinol, intraperitoneally injected into the animals, was incorporated into the outer and inner segments of retinal photoreceptors, but not into the pineal organ. It is concluded that the mouse pineal organ contains the authentic apoprotein of rhodopsin but that it lacks retinal derivatives as essential components of all known vertebrate photopigments. Consequently, the “photoreceptor-specific” proteins of the mammalian pineal organ are not involved in photoreception and phototransduction, but may serve other functions to be explored in future studies.

Morphological and immunocytochemical heterogeneity of cultured pinealocytes from one‐week‐and two‐month‐old rats: Planimetric and densitometric investigations

Abstract: In vitro preparations of rat pinealocytes are widely used for biochemical analyses of signal transduction processes. This paper deals with morphological and immunocytochemical features of such preparations. Special attention was paid to the problems of whether pinealocytes represent a heterogeneous cell population and how such heterogeneity may develop during ontogeny. The investigations were performed with cells which were obtained from the pineal organ of one-week- and two-month-old rats, attached to synthetic peptide-coated coverslips or tissue culture chamber slides, and maintained under in vitro conditions overnight. The attached cells were then fixed with paraformaldehyde. These preparations yielded monolayers of spherical cells of different sizes; most cells were isolated, but some of them were aggregated and formed small clusters. On the average, the cells from the one-week-old animals were smaller than the cells from the two-month-old animals. Immunocytochemical demonstration of S-antigen, a pinealocyte-specific marker, showed that the majority of the cells from two-month-old animals were intensely or moderately labelled. Pinealocytes from one-week-old animals were less S-antigen immunoreactive. Only very few cells (less than 1/1000 displayed glial fibrillary acidic protein (GFAP)-immunoreactivity. Planimetric investigations of the cell size and semiquantitative densitometric investigations of the intensity of the S-antigen immunoreaction revealed that (i) pinealocytes kept in vitro form a heterogeneous cell population, and that (ii) this heterogeneity increased during postnatal development from one-week-old to two-month-old animals. Two groups of pinealocytes can be distinguished based on their developmental fate: pinealocytes of one group grow dramatically, but show only a moderate increase in S-antigen immunoreactivity, and pinealocytes of the other group retain their size, but display a distinct increment in S-antigen immunoreactivity.

Cyclic GMP-activated channels of the chick pineal gland: effects of divalent cations, pH, and cyclic AMP.

Abstract: Chick pineal cells maintained in dissociated cell culture express an intrinsic photosensitive circadian oscillator, but the mechanisms of phototransduction in avian pinealocytes are not fully understood. In this study, we have used inside-out patches to examine the characteristics of cyclic GMP-activated channels of chick pinealocytes in more detail, concentrating on the effects of factors known to modulate the secretion of melatonin and/or the function of circadian pacemakers. In most patches, the predominant conductance state was 19 pS in symmetrical 145 mM NaCl. But in some patches, a second cyclic GMP-activated channel with a unitary conductance of 29 pS was also present. The current flowing through cyclic GMP-activated channels was not affected by application of salines containing 1 μM Ca2+ to the cytoplasmic face of the patch membrane. By contrast, application of 1 mM Ca2+ caused a partial reduction in cyclic GMP-activated current at all membrane potentials. Application of 1–5 mM Mg2+ ions caused a virtually complete blockade of current at positive membrane potentials, but caused only a small decrease in current at negative membrane potentials. No obvious differences in the gating of cyclic GMP-activated channels were observed in pH 8.2, 7.4 or 6.2 salines. Application of salines containing 100 μM, 500 μM, or 1 mM cyclic AMP did not cause activation of the channels, but 5 mM cyclic AMP evoked a low level of channel activity. Application of 5 mM but not 100 μM cyclic AMP decreased the probability of channel activation caused by 20–100 μM cyclic GMP and also increased the percentage of openings to an 11 pS subconductance state. Thus, cyclic AMP acts as a weak partial agonist. Nevertheless, the gating of these channels does not seem to be controlled directly by physiologically relevant changes in intracellular Ca2+, pH, or cyclic AMP.

Experimental autoimmune pinealitis in the rat : ultrastructure and quantitative immunocytochemical characterization of mononuclear infiltrate and MHC class II expression

Abstract: Lewis rats immunized with Peptide M (an oligopeptide epitope of the S-antigen protein) developed experimental autoimmune uveoretinitis (EAU) and experimental autoimmune pinealitis (EAP). Temporal changes in mononuclear infiltrate to the pineal gland were quantitated by computer image analysis of sections immunostained with monoclonal antibodies to specific mononuclear populations. T helper/inducer cells (W3/25+) and monocyte/macrophages (OX-42 +) were elevated during the early phases of inflammation (day 15) while cytotoxic/suppressor T cells (OX-8 +) were elevated at days 15 and 21. Expression of MHC class II (OX-6) was markedly enhanced on pineal glia, but was not present on vascular endothelia during EAP. Ultrastructurally, many capillaries exhibited thickenings of the endothelia and basal lamina. EAP had little effect on the fine structure of pinealocytes and glia and there was little evidence of cellular destruction by day 21, in contrast to the extensive retinal destruction resulting from EAU. These...

Ultrastructural study of post-hatching development in the pineal gland of the Japanese quail.

Abstract: The pineal gland of the Japanese quail (Coturnix coturnix japonica) retained numerous follicular structures, each of which was composed mostly of pinealocytes and supporting cells radially arranged around the follicular lumen during the first 6 weeks of age. In the pinealocytes, paraboloids and regular stacks of lamellar disks, which were demonstrated at 1 day of age in a previous study [14], rapidly decreased in number after 1 week of age. From 1 to 6 weeks of age, the pinealocytes had more developed lamellar whorls in the follicular lumen with the increased numbers of mitochondria in their apical protrusions and synaptic ribbons in their basal processes. In addition to these photosensory elements, pigment-containing cells occasionally accumulated in the basal side of the follicular structure. Their pigment granules, which were considered as melanin, gradually became larger and more roundish in shape after 1 week of age. The histological elements associated with the secretory function of the quail pineal were dense-cored vesicles and lysosomes in the pinealocytes and dense bodies in the supporting cells. The synaptic ribbons and the dense-cored vesicles in the pinealocytes rapidly increased in number at 1 week of age, scarcely exhibiting distinct changes thereafter. Thus, it is clarified that the foregoing photosensory and secretory elements change during the post-hatching development. Furthermore, it is suggested that, in respect of these elements, the pineal gland of the quail assumes more active appearances than that of the chicken.

An immunohistochemical study of neuropeptide Y in the bovine pineal gland

Abstract: An immunohistochemical study of the bovine pineal gland was performed using rabbit polyclonal antibodies raised against neuropeptide Y (NPY) or against the C-terminal flanking peptide of proNPY (CPON). A large number of NPY/CPON-immunoreactive (IR) nerve fibers were demonstrated throughout bovine pineal gland. The IR-fibers were located in the capsule of the gland, usually piercing into the gland together with blood vessels. In the gland itself, the fibers wen also located intraparenchymally between the pinealocytes. Within the rostral and caudal areas of the pineal stalk, NPY-IR fibers were also observed, and these fibers could be followed not only into the gland bul also to the habenular and posterior commissures. The morphological localization of the NPY-IR nerve fibers in the bovine pineal gland indicate that the majority of fibers originate from the superior cervical ganglion. However, some fibers probably originate from the brain itself.

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.

Abstract: 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.

Development of the photoreceptor outer segment-like cilia of the CSF-contacting pinealocytes of the ferret (Putorius furo).

Abstract: The postnatal development of the pineal organ of the ferret (Putorius furo) was investigated electron-microscopically with special interest given to the cerebrospinal fluid (CSF)-contacting pinealocytes and their large, vesiculated cilia. In the pineal of the newborn ferrets, there is a lumen--a pineal ventricle--which is a diverticle of the third ventricle of the diencephalon. The luminal surface of the pineal is bordered by ependymal cells and CSF-contacting pinealocytes. A sensory, 9 x 2 + 0 type cilium arises from the free surface of the pinealocytes and thickens in the first week. There are mitotic figures in the wall of the pineal ventricle, being reduced to a pineal recess during the second and third postnatal week. In two week-old animals, vesicles appear in the cilia of the pinealocytes. The vesicles may form rows and fill the enlarged cilium at the third week. Near the basal bodies, a proximal connecting piece remains narrow and free of vesicles. In older animals, there are multivesicular and dense bodies in the pineal cilia. The reduction of the pineal ventricle closes the CSF-contacting cilia in the intercellular spaces. Axon-like processes of pinealocytes form synaptic ribbon-containing terminals on secondary pineal neurons. Axons of pineal neurons enter the fiber bundles of the pineal tract running to the habenular nuclei. All these structures do not differ from the light conducting pathway of the submammalian pineals. The ultrastructure of the cilia investigated resembles that of the developing outer segments of the retina and represents a preserved light perceiving structure of the mammalian pinealocytes. Further studies are necessary to elucidate whether the early differentiation of the cilia and synapses indicates a timing of the circadian light rhythmicity in young ferrets by direct pineal photosensitivity.

Expression of somatostatin in rat pineal cells in culture

Abstract: The expression of somatostatin mRNA was investigated in rat pineal cells after 1 week in culture, using reverse transcription of mRNA into cDNA and the polymerase chain reaction. The positive expression in cultured pineal cells demonstrates the capacity of this gland to synthesize somatostatin in denervated cells. Thus, apart from the neural origin of pineal somatostatin, which has been described in detail in the bovine species, a parenchymal source is demonstrated.