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Rhythmic activity of arylalkylamine N-acetyltransferase (AANAT) determines melatonin synthesis in rat pineal gland. The transcriptional regulation of AANAT involves the activating and inhibiting transcription factors of the cyclic AMP (cAMP)-signaling pathway, cAMP response element-binding protein and inducible cAMP early repressor (ICER), respectively. Activation of this pathway is centered around norepinephrine, stimulating beta(1)-adrenergic receptors, but various other transmitters can modulate melatonin biosynthesis. To compare the transcriptional impact of norepinephrine with that of other neurotransmitters on melatonin synthesis, we determined ICER protein levels in pinealocytes and, in parallel, hormone secretion. The dose-dependent inductions of ICER protein by norepinephrine, the beta(1)-adrenergic receptor agonist isoproterenol, vasoactive intestinal peptide, pituitary adenylate cyclase-activating polypeptide, and adenosine are correlated to regulatory dynamics in melatonin production. Importantly, ICER protein induction required lower ligand concentrations than the induction of melatonin biosynthesis. Although neuropeptide Y, glutamate, and vasopressin altered norepinephrine-stimulated hormone production without affecting ICER levels, the activation of voltage-gated cation channels increased ICER without affecting hormone synthesis. Sensitivity and versatility of ICER induction in pinealocytes make these neuroendocrine cells a valuable model system in which to study molecular interactions determining a regulated gene expression.

Summary: Calcium plays an important role for signal transduction in the mammalian pineal organ. The regulation of the intracellular concentration of free calcium probably involves calcium-binding proteins of the calmodulin superfamily. In the present study, we have investigated the expression of calretinin, one member of this superfamily, in the pineal organ of hamsters, gerbils and guinea-pigs by means of immunochemical and immunocytochemical analyses with a calretinin-specific antiserum. In immunoblots this antibody recognized a single protein band of sim29 kDa in the brain and pineal organ of all three mammalian species. Immunocytochemical investigations of serial semithin sections of plastic-embedded pineals revealed the constant occurrence of variable numbers of calretinin-positive cells throughout all glands. In order to identify the immunopositive cells precisely, adjacent sections were exposed to antibodies against various marker proteins of pineal cell types, i.e., synaptophysin, neuron-specific enolase, protein gene product 9.5, S-antigen, vimentin and S-100. By this approach, calretinin could be localized to vimentin-positive cells in the gerbil which are generally considered as interstitial glial cells. Likewise, calretinin-positive cells in the guinea-pig probably correspond to interstitial cells, taking into account their morphology and the lack of calretinin immunoreactivity in pinealocytes. The unusual expression of calretinin in astrocyte-like cells further supports the notion that pineal glial cells are endowed with peculiar properties. In contrast to gerbil and guinea-pig, a subpopulation of pinealocytes displayed calretinin immunoreactivity in the hamster. This finding adds to the hypothesis that in pinealocytes of some species calretinin plays a role in calcium-mediated signal transduction which eventually is linked to melatonin synthesis. Our results demonstrate that calretinin is a regular constituent of pineal glands in three mammalian species, but that its cellular localisation shows interspecific variation. This variation suggests that the protein is involved in diverse calcium-mediated functions in the mammalian pineal gland.

Summary This study attempts to reconstruct the early phylogenetic history of macroglial cells among craniates. Since glia does not fossilize, such a reconstruction must be based on a cladistic comparison of glial characters in the Recent craniate taxa (hagfishes, lampreys, and gnathostomes); however, there are only few data on glial morphology and none on glial immunocytochemistry in hagfishes. Therefore, we investigated the presence and localization of various macroglia-specific epitopes in the brain and spinal cord of the Pacific hagfish,Eptatretus stouti (Myxinoidea) by means of immunocytochemistry. Antibodies directed against S100-pfotein and vimentin showed no cross reactivity. Antibodies directed against glial fibrillary acidic protein and glutamine synthetase labelled various glial structures. Glial fibrillary acidic protein-like immunoreactivity was observed in ependymal cells with radially oriented processes in some regions. However, throughout the entire CNS, labelling of non-ependymal cells and their processes prevailed. The processes of these cells made occasional vascular contacts and they also made contacts with neuronal perikarya. Glutamine synthetase-like immunoreactivity was also found in some processes with radial orientation and in ependymal cells; but the antibody stained mainly non-ependymal cells which gave rise to a felt-like meshwork of interdigitating fine and very fine processes penetrating the neuropil of the entire brain. Additionally, there was labelling in the walls of blood vessels and in processes enwrapping individual neurons. The occurrence of glial fibrillary acidic protein- and glutamine synthetase-like immunoreactivity in non-ependymal glial elements in the brain of hagfishes and the relative scarcity of labelling in radial glial elements necessitates a re-interpretation of the evolutionary history of glial cells. Non-ependymal macroglia with immunocytochemical and morphological characters resembling typical (mammalian) astrocytes appears to be as primitive as the various forms of radial ependymal glia.

1. Membrane voltage (Vm) recordings were obtained from isolated rat pinealocytes using the patch‐clamp technique. In parallel to the electrophysiological experiments, intracellular Ca2+ measurements were performed using fura‐2. 2. The resting Vm averaged ‐43 mV and replacement of extracellular NaCl by KCl completely depolarized the cells. This indicates that the resting Vm is dominated by a K+ conductance. Single‐channel recordings revealed the presence of a large conductance Ca(2+)‐activated charybdotoxin‐sensitive K+ channel. 3. Application of ACh (100 microM) depolarized the pinealocytes on average by 16 mV. The depolarizing effect of ACh was mimicked by nicotine (50 microM) and was prevented by tubocurarine (100 microM). 4. The ACh‐induced depolarization was largely abolished in the absence of extracellular Na+, but was not significantly affected by extracellular Ca2+ removal. 5. Application of ACh (100 microM) caused an increase in [Ca2+]i. This increase was completely dependent on the presence of extracellular Ca2+ and was largely reduced after extracellular Na+ removal. Nifedipine (1 microM) reduced the ACh‐induced increase in [Ca2+]i by about 50%. 6. Our findings indicate that in rat pinealocytes stimulation of a nicotinic ACh receptor (nAChR) induces depolarization mainly by Na+ influx via the nAChR. The depolarization then activates L‐type Ca2+ channels, which are responsible for the nifedipine‐sensitive portion of the intracellular Ca2+ increase. Ca2+ influx via the nAChR probably also contributes to the observed rise in [Ca2+]i.

Abstract Considerable progress is currently being made in elucidating the molecular basis of the circadian (photoneuroendocrine) system by use of transgenic mice generated from the inbred strains C57BL and C3H. As in all other vertebrate species, the pineal organ is an important component of the photoneuroendocrine system in these mouse strains, but very little is known about its morphological and immunocytochemical features. We therefore investigated the pineal organ and the adjacent epithalamic region of adult, 10-, and 5-day-old C57BL and C3H mice for S-antigen, serotonin, and dopamine-ß-hydroxylase (DBH) immunoreactions. In adult animals, the pineal organ was more than 2 times bigger in C3H than in C57BL mice. In younger animals, this difference was already evident, but less pronounced. The S-antigen immunoreactivity was more intense in adult C3H than in C57BL mice. This difference developed with increasing age; it was not yet detectable in 5-day-old animals. The intensity of the serotonin immunoreaction was similar in both strains at all stages investigated. However, the serotonin immunoreaction was more pronounced in adult than in young animals. The relative DBH-immunoreactive area (used as a marker for the sympathetic innervation of the pineal organ) was much bigger in C3H than in C57BL mice; within each strain it remained relatively constant during postnatal development. Adult individuals of both strains contained S-antigen- and serotonin-immunoreactive cells in the habenular complex. Their number increased with age, but they were always more numerous in C3H. In conclusion, the study has shown considerable differences in pineal morphology between C3H and C57BL, which may be related to the well-known differen- ces in melatonin formation between these two strains.

Abstract:  Immunocytochemical double-labeling methods are important tools in cell and neurobiology. Here we describe a method which is based on double immunofluorescence and allows specific detection of two different antigens located in the same cell compartment by two primary antibodies raised in the same species. As an example, we present the double-immunolabeling method for the S-antigen (SAg), a photoreceptor-specific protein, and the indoleamine serotonin (5HT) in dissociated trout and rat pineal cells immobilized on coversliped and in frozen sections of the trout pineal organ. As a first step, the preparations on the slides or coverslips were sequentially incubated with the first primary antibody (rabbit anti-SAg), the fluorescein-labeled (anti-rabbit) secondary antibody, and then with normal rabbit serum. Meanwhile, the second primary antibody (rabbit anti-5HT) was coupled to a Cy3-labeled secondary (anti-rabbit) antibody in a reaction tube and excess binding sites were quenched with normal rabbit serum. This complex was applied to the specimens after completion of the first (SAg) immunoreaction on the slide. For control experiments, the first (anti-SAg) or the second (anti-5HT) primary antibody were omitted. Most of the rat and trout pinealocytes were double immunolabeled for SAg and 5HT. In the trout, few cells contained SAg or 5HT immunoreaction only. This underlines the selectivity of each immunoreaction. The results show that the method can be used for the analysis of whole cells and tissue sections by means of conventional fluorescence and confocal laser scanning microscopy.

The problem of the blood-brain barrier in the pineal organ of the rainbow trout, Salmo gairdneri, was investigated following intraperitoneal or intracardial injections of several tracers and dyes with different molecular weights. As demonstrated at the light-microscopic level, repeated injections of trypan blue or horseradish peroxidase (HRP) resulted in an accumulation of these substances in the pineal epithelium (parenchyma). By use of the electron microscope, HRP was found in electron-dense bodies, probably lysosomes, in (i) the endothelial cells and perivascular macrophages 4 h after intraperitoneal injection, (ii) the supporting cells and intrapineal or luminal macrophages 8 h after injection, and (iii) the receptor cells 24 h after injection of the tracer. Ferritin particles penetrated the fenestrated endothelium of pineal capillaries. They were confined to vesicles, vacuoles and the smooth endoplasmic reticulum of the supporting cells as well as to the synaptic vesicles and the smooth endoplasmic reticulum of the pineal photoreceptors. The intercellular passage of tannic acid mixed with the fixative was blocked at the luminal junctional complex separating the pineal lumen from the basal portion of the pineal epithelium. The passive intercellular transport of substances with high molecular weight from the bloodstream to the cerebrospinal-fluid compartment is thus prevented. However, no blood-brain barrier exists for exogenously administered proteins, which are rapidly taken up by pineal cells and actively transported in a transcellular manner.

By use of the indirect peroxidase-antiperoxidase immunohistochemical technique, nerve terminals exhibiting vasoactive intestinal polypeptide (VIP)-like-immunoreactivity were demonstrated in the pineal gland of the Mongolian gerbil (Meriones unguiculatus).

The angioarchitecture of the pineal organ of the rainbow trout (Salmo gairdneri) was investigated by means of the corrosion-cast preparation method and scanning electron microscopy. Two main arteries (aa. epiphyseales) supply the pineal parenchyma. They emerge from the aa. cerebri anteriores and run in the fissure between the prosencephalon and the mesencephalon. After entering the pineal stalk, the aa. epiphyseales branch off into several arterioles, most of which extend to the pineal end-vesicle where they give rise to a lobular, bilaterally symmetric capillary network. Capillaries establishing the main portion of the pineal vessels appear widened in comparison to those supplying other portions of the brain and resemble capillaries in other endocrine organs. In Salmo gairdneri, no specialized system of portal vessels appears to exist between the pineal organ and other portions of the brain.