Horst-W. Korf

The Pineal Organ as a Component of the Biological Clock

In conclusion, several trends are observed in regard to the phylogenetic development of the pineal organ, which are relevant for our understanding of the evolution of biological clock mechanisms. 1. The pineal organ of all vertebrates investigated thus far is capable of producing and releasing melatonin. Melatonin is rhythmically produced and released during darkness and, thus, represents an important neuroendocrine information on the ambient photoperiod. 2. The rhythmic production of melatonin is under control of endogenous oscillators and photoreceptor cells. In several nonmammalian species, these endogenous oscillators and photoreceptors are located within the pineal organ itself. In some avian species, the inherent rhythmicity of the pineal organ appears to be influenced by pacemakers located in other parts of the central nervous system. Their information may be transmitted to the pineal organ via the sympathetic innervation. This innervation develops progressively in the course of phylogeny. In mammals certain pinealocytes express proteins which are specific of retinal and pineal photoreceptors, but these proteins are obviously not involved in photoreception and phototransduction. The mammalian pineal organ lacks not only functioning photoreceptors, but also endogenous oscillators. The photoreceptor cells involved in regulation of the melatonin biosynthesis are located in the retina; the major endogenous oscillator is the suprachiasmatic nucleus (SCN) of the hypothalamus. Information from the retina and the SCN is transmitted to the mammalian pineal organ via a complex neuronal chain, whose last member is the sympathetic innervation originating from the superior cervical ganglion. This innervation is mandatory to maintain the rhythm of the melatonin biosynthesis in the mammalian pineal organ. Interestingly, the effects of noradrenaline, the major neurotransmitter in the sympathetic nerve fibers, displays opposite effects on the melatonin biosynthesis in birds and mammals: it stimulates the melatonin biosynthesis in the mammalian pineal organ, but inhibits the melatonin formation in the chicken. This conversion occurs at the level of the adrenoreceptors. 3. The intrapineal nerve cells giving rise to pinealofugal neuronal projections are reduced in the course of phylogeny. Nevertheless, direct neuronlike connections appear to exist between the pineal organ and the central nervous system of mammals. These projections originate from a population of pinealocytes. Whether such projections are involved in biological clock mechanisms remains an issue not yet resolved. The ontogenetic data reviewed support the notion that, in lower vertebrates, melatonin biosynthesis is primarily controlled by intrapineal photoreceptors, whereas, in mammals, it depends on retinal photoreceptors and the sympathetic innervation of the pineal.(ABSTRACT TRUNCATED AT 400 WORDS)

The pituitary adenylate cyclase-activating polypeptide-induced phosphorylation of the transcription factor CREB (cAMP response element binding protein) in the rat suprachiasmatic nucleus is inhibited by melatonin

The mammalian hypothalamic suprachiasmatic nucleus (SCN) is an endogenous pacemaker generating circadian rhythms. SCN activity is synchronized with environmental light/dark cycles by photic information primarily transmitted via the retinohypothalamic tract (RHT). The SCN controls synthesis and release of melatonin, the hormone of the pineal gland. Melatonin itself feeds back to the SCN. Using brain slice technique and immunocytochemistry we demonstrate that (1) pituitary adenylate cyclase-activating polypeptide (PACAP) induces the phosphorylation of the transcription factor cAMP response element binding protein (CREB) in the SCN during late subjective day and (2) melatonin inhibits this PACAP-induced phosphorylation. Our data suggest that PACAP is a neurotransmitter which affects gene expression in the SCN probably via the cAMP signaling pathway and that the antagonistic effect of melatonin mirrors a feed-back loop within the circadian system.

Stimulation of a nicotinic ACh receptor causes depolarization and activation of L‐type Ca2+ channels in rat pinealocytes.

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.

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

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

Signal transduction molecules in the rat pineal organ: Ca2+, pCREB, and ICER.

The mammalian pineal organ transduces light-dependent neural inputs into a hormonal output. This photoneuroendocrine transduction results in a largely elevated concentration of the pineal hormone melatonin at night. The rhythm in melatonin production and secretion depends on activation and inactivation of transcriptional, translational, and posttranslational mechanisms fundamentally linked to two second messenger systems, the cAMP- and the Ca(2+)-signal transduction pathways. Here we review molecular biological, immunocytochemical, and single-cell imaging studies, which demonstrate a time- and substance-specific activation of these signaling pathways in rat pinealocytes. The data provide a framework for understanding the complex interactions between second messengers (cAMP, Ca2+), transcription factors (CREB, ICER), and their role in regulation of melatonin synthesis. The data have proven the rat pinealocyte to be an interesting model to study transmembrane signaling pathways which may be common to both neuroendocrine and neuronal cells.

Single-cell [Ca2+]i analysis and biochemical characterization of pinealocytes immobilized with novel attachment peptide preparation.

Single-cell image analysis of rat pinealocytes has been difficult because they do not attach readily to coated or uncoated surfaces and typically adhere in clusters to fibroblast-like cells. In the present report, a new method for the rapid attachment of rat pinealocytes is described. Cells were prepared using papain digestion and density centrifugation and then were placed on coverslips or slides coated with PepTite-2000, a preparation containing the attachment peptide sequence Arg-Gly-Asp. Cells immobilized with this preparation responded to norepinephrine treatment with an increase in cyclic AMP and melatonin production. Single-cell analysis of Fura-2-loaded cells revealed that norepinephrine increased [Ca2+]i. This development makes it possible to conduct routine single-cell image analysis and other studies of freshly isolated rat pinealocytes.

Melatonin Receptors in the Spinal Cord

The pineal hormone, melatonin, plays an important role in the regulation of diurnal and seasonal rhythms in animals. In addition to the well established actions on the brain, the possibility of a direct melatonin action on the spinal cord has to be considered. In our laboratory, we have obtained data suggesting that melatonin receptors are present in the spinal cords of birds and mammals. Using radioreceptor binding and quantitative autoradiography assays with 2-[125I]iodomelatonin as the specific melatonin agonist, melatonin binding sites have been demonstrated in the rabbit and chicken spinal cords. These sites are saturable, reversible, specific, guanosine nucleotide-sensitive, of picomolar affinity and femtomolar density. The linearity of Scatchard plots of saturation data and the unity of Hill coefficients indicate that a single class of melatonin binding sites is present in the spinal cord membranes studied. The picomolar affinity of these sites is in line with the circulating levels of melatonin in these animals suggesting that these sites are physiologically relevant. Autoradiography studies in the rabbit spinal cord show that melatonin binding sites are localized in the central gray substance (lamina X). In the chicken spinal cord, these binding sites are localized in dorsal gray horns (laminae I-V) and lamina X. As lamina X and laminae I-II have similar functions, melatonin may have comparable roles in the chicken and rabbit spinal cords. Moreover, in the chicken spinal cord, the density of 2-[125I]iodomelatonin binding in the lumbar segment was significantly higher than those of the cervical and thoracic segments. The densities of these binding sites changed with environmental manipulations. When chickens were adapted to a 12L/12D photoperiod and sacrificed at mid-light and mid-dark, there was a significant diurnal variation in the density (maximum number of binding sites; Bmax) of melatonin binding sites in the spinal cord. After constant light treatment or pinealectomy, the Bmax of melatonin receptors in the chicken spinal cord increased significantly in the subjective mid-dark period. Moreover, there was an age-related decrease in the 2-[125I]iodomelatonin binding to the chicken spinal cord. Our results suggest that melatonin receptors in the chicken spinal cord are regulated by environmental lighting and change with development. These receptors may play an important role in the chronobiology of spinal cord function. The biological responses of melatonin on spinal cords have also been demonstrated in vitro. Melatonin decreased the forskolin-stimulated cAMP production in the chicken spinal cord explant. Preincubation with pertussis toxin blocked the melatonin effect. Our results suggest that melatonin receptors in the chicken spinal cord are linked to the adenylate cyclase via a pertussis toxin-sensitive G protein and that melatonin binding sites in spinal cords are melatonin receptors with biological functions. These receptors may be involved in the regulation of spinal cord functions related to sensory transmission, visceral reflexes and autonomic activities.

Confocal laser scanning and electron-microscopic analyses of the relationship between VIP-like and GnRH-like-immunoreactive neurons in the lateral septal-preoptic area of the pigeon

Abstract The lateral septum and the preoptic area of birds comprise neurons immunoreactive (ir) for vasoactive intestinal polypeptide (VIP) and gonadotropin-releasing hormone (GnRH). By use of immunohistochemical single- and double-labeling techniques, we have investigated the distribution and the connections of these two types of peptidergic neurons in the lateral septal-preoptic area of the pigeon at both the light- and electron-microscopic levels. An accumulation of VIP-like-ir neurons, some of which are cerebrospinal fluid-contacting neurons, is found in the area adjacent to the ventromedial walls of the lateral ventricles in the lateral septum corresponding to the medial part of the lateral septal organ. VIP-like-ir terminals are scattered throughout the lateral septal-preoptic area, which also contains GnRH-like-ir cell bodies. The number of GnRH-like-ir cell bodies in the lateral septum is smaller than that of the VIP-like-ir neurons. GnRH-like-ir cells have a simple bipolar or multipolar shape and a beaded axon that emerges from the soma or one of the proximal dendrites. Confocal laser scanning microscopy has shown VIP-like-ir terminals in close apposition to GnRH-like-ir cell bodies in the lateral septal-preoptic area. Furthermore, the electron-microscopic double-immunolabeling has revealed synaptic contacts between VIP-like-ir axon terminals and GnRH-like-ir cell bodies or dendrites. These contacts, however, do not show synaptic specializations. The present results suggest that functional interactions take place between VIP and GnRH neurons in the lateral septal-preoptic area of the pigeon and that these interactions are involved in mediating photoperiodic responses.

Electron-microscopic investigations of vasoactive intestinal peptide (VIP)-like immunoreactive terminal formations in the lateral septum of the pigeon.

Vasoactive intestinal peptide (VIP)-like immunoreactive terminal fields were examined in the lateral septum of the pigeon by means of immunocytochemistry. According to light-microscopic observations, these projections originated from VIP-like immunoreactive cerebrospinal fluid (CSF)-contacting neurons, which are located in the ependymal layer of the lateral septum and form a part of the lateral septal organ. The processes of these cells gave rise to dense terminal-like structures in the lateral septum. Pre-embedding immuno-electron microscopy revealed that VIP-like immunoreactive axon terminals had synaptoid contacts with perikarya of small VIP-immunonegative neurons of the lateral septum, which were characterized by an invaginated nucleus, numerous mitochondria, a well-developed Golgi apparatus, endoplasmic reticulum and a small number of dense-core vesicles (about 100 nm in diameter). VIP-like immunoreactive axons were also seen in contact with immunonegative dendrites in the lateral septum. In both axosomatic and axodendritic connections, VIP-like immunoreactive presynaptic terminals contained large dense-core vesicles, clusters of small vesicles and mitochondria. These findings suggest that VIP-immunoreactive neurons of the lateral septal organ project to small, presumably peptidergic nerve cells of the lateral septum and that the VIP-like neuropeptide serves as a neuromodulator (-transmitter) in this area.

Effects of neuroactive substances on the activity of subcommissural organ cells in dispersed cell and explant cultures

Abstract. The subcommissural organ (SCO), an ependymal (glial) circumventricular organ, releases glycoproteins into the cerebrospinal fluid; however, the regulation of its secretory activity is largely unknown. To identify neuroactive substances that may regulate SCO activity, we investigated immunocytochemically identified bovine SCO cells by means of calcium imaging. This analysis was focused on: (1) serotonin (5HT) and substance P (SP), immunocytochemically shown to be present in axons innervating the bovine SCO; and (2) ATP, known to activate glial cells. 5HT had no effect on the intracellular calcium concentration ([Ca2+]i), and its precise role remains to be clarified. SP elicited rises in [Ca2+]i in approx. 30% and ATP in even 85% of the analyzed SCO cells. These effects were dose-dependent, involved NK3 and P2Y2 receptors linked to G protein and phospholipase C (PLC) activation, and could not be mimicked by forskolin or 8-bromo-cAMP. In 50% of the SP-sensitive cells, the increases in [Ca2+]i comprised calcium release from thapsigargin-sensitive intracellular stores and an influx of extracellular calcium via protein kinase C (PKC)-induced opening of L-type voltage-gated calcium channels (VGCCs). In the remaining SP-sensitive cells, the increase in [Ca2+]i was caused exclusively by influx of extracellular calcium via VGCCs of the L-type. In all ATP-sensitive cells the increase in [Ca2+]i involved calcium release from thapsigargin-sensitive intracellular stores and a PKC-mediated influx of extracellular calcium via L-type VGCCs. Our data suggest that SP and ATP are involved in regulation of the activity of SCO cells.