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A local renin-angiotensin system (RAS) has been postulated in the pineal gland. In addition to angiotensin II (Ang II), other active metabolites have been described. In this study, we aimed to investigate a role for Ang IV in melatonin synthesis and the presence of its proposed (IRAP)/AT4 receptor (insulin-regulated aminopeptidase) in the pineal gland. The effect of Ang IV on melatonin synthesis was investigated in vitro using isolated pinealocytes. IRAP protein expression and activity were evaluated by Western blot and fluorimetry using Leu-4Me-β-naphthylamide as a substrate. Melatonin was analyzed by HPLC, calcium content by confocal microscopy and cAMP by immunoassay. Ang IV significantly augmented the NE-induced melatonin synthesis to a similar degree as that achieved by Ang II. This Ang IV effect in pinealocytes appears to be mediated by an increase in the intracellular calcium content but not by cAMP. The (IRAP)/AT4 expression and activity were identified in the pineal gland, which were significantly higher in membrane fractions than in soluble fractions. Ang IV significantly reduced IRAP activity in the pineal membrane fractions. The main findings of the present study are as follows: (1) Ang IV potentiates NE-stimulated melatonin production in pinealocytes, (2) the (IRAP)/AT4 receptor is present in the rat pineal gland, and (3) Ang IV inhibits IRAP activity and increases pinealocytes [Ca2+]i. We conclude that Ang IV is an important component of RAS and modulates melatonin synthesis in the rat pineal gland.

Survivors of hypoxic-ischemic brain damage (HIBD), besides impairment of psychomotor development, often develop circadian rhythm disorders, although the underlying mechanisms are largely unknown. Here, we first verified that mRNA and protein expression of pineal aralkylamine N-acetyltransferase (Aanat), a key regulator for melatonin (MT) synthesis, along with MT, were severely impaired after HIBD. In addition, we demonstrated that neonatal HIBD disrupted the circadian rhythmicity of locomotor activities in juvenile rats. Based on bioinformatics analysis of a high throughput screening of miRNA expression changes after HIBD (Ding et al., 2015), we identified one microRNA, miR-325-3p, as a potential candidate responsible for the down regulation of Aanat after HIBD. Luciferase reporter assays demonstrated a specific interaction between miR-325-3p and Aanat mRNA 3'-UTR. miR-325-3p blocked norepinephrine (NE) induced Aanat activation in cultured pinealocytes. In addition, miR-325-3p inhibition partially rescued Aanat induction by NE, which was significantly reduced under oxygen glucose deprivation. By elucidating the role of pineal miR-325-3p on Aanat expression upon injury, our study provides new insights into the pathophysiological mechanisms of circadian dysfunction and potential therapeutic targets after HIBD.

The zebrafish pineal gland plays an important role in different physiological functions including the regulation of the circadian clock. In the fish pineal gland the pinealocytes are made up of different segments: outer segment, inner segment and basal pole. Particularly, in the outer segment the rhodopsin participates in the external environment light reception that represents the first biochemical step in the melatonin production. It is well known that the rhodopsin in the adult zebrafish is well expressed in the pineal gland but both the expression and the cellular localization of this protein during development remain still unclear. In this study using qRT-PCR, sequencing and immunohistochemistry the expression as well as the protein localization of the rhodopsin in the zebrafish from larval (10 dpf) to adult stage (90 dpf) were demonstrated. The rhodopsin mRNA expression presents a peak of expression at 10 dpf, a further reduction to 50 dpf before increasing again in the adult stage. Moreover, the cellular localization of the rhodopsin-like protein was always localized in the pinealocyte at all ages examined. Our results demonstrated the involvement of the rhodopsin in the zebrafish pineal gland physiology particularly in the light capture during the zebrafish lifespan.

Entrainment of circadian clocks to environmental cues such as photoperiod ensures that daily biological rhythms stay in synchronization with the Earth's rotation. The vertebrate pineal organ has a conserved role in circadian regulation as the primary source of the nocturnal hormone melatonin. In lower vertebrates, the pineal has an endogenous circadian clock as well as photoreceptive cells that regulate this clock. The zebrafish opsin protein Exo-rhodopsin (Exorh) is expressed in pineal photoreceptors and is a candidate to mediate the effects of environmental light on pineal rhythms and melatonin synthesis. We demonstrate that Exorh has an important role in regulating gene transcription within the pineal. In developing embryos that lack Exorh, expression of the exorh gene itself and of the melatonin synthesis gene serotonin N-acetyl transferase 2 (aanat2) are significantly reduced. This suggests that the Exorh protein at the cell membrane is part of a signaling pathway that positively regulates transcription of these genes, and ultimately melatonin production, in the pineal. Like many other opsin genes, exorh is expressed with a daily rhythm: mRNA levels are higher at night than during the day. We found that the transcription factor Orthodenticle homeobox 5 (Otx5) activates exorh transcription, while the putative circadian clock component Period 3 (Per3) represses expression during the day, thereby contributing to the rhythm of transcription. This work identifies novel roles for Exorh and Per3, and gives insight into potential interactions between the sensory and circadian systems within the pineal.

The photoreceptive retina and the non-photoreceptive pineal gland are components of the circadian and the melatonin forming system in mammals. To contribute to our understanding of the functional integrity of the circadian system and the melatonin forming system we have compared the daily oscillation of the two tissues under various seasonal lighting conditions. For this purpose, the 24-h profiles of the expression of the genes coding for arylalkylamine N-acetyltransferase (AA-NAT), nerve growth factor inducible gene-A (NGFI-A), nerve growth factor inducible gene-B (NGFI-B), retinoic acid related orphan receptor β (RORβ), dopamine D4 receptor, and period2 (Per2) have been simultaneously recorded in the retina and the pineal gland of rats under short day (light/dark 8:16) and long day (light/dark 16:8) conditions. We have found that the cyclical patterns of all genes are phase-advanced in the retina, often with a lengthened temporal interval under short day conditions. In both tissues, the AA-NAT gene expression represents an indication of the output of the relevant pacemakers. The temporal phasing in the AA-NAT transcript amount between the retina and the pineal gland is retained under constant darkness suggesting that the intrinsic self-cycling clock of the retina oscillates in a phase-advanced manner with respect to the self-cycling clock in the suprachiasmatic nucleus, which controls the pineal gland. We therefore conclude that daily rhythms in gene expression in the retina are phase-advanced with respect to the pineal gland, and that the same temporal relationship appears to be valid for the self-cycling clocks influencing the tissues.

Chronic stress affects brain areas involved in learning and emotional responses. These alterations have been related with the development of cognitive deficits in major depression. Moreover, stress induces deleterious actions on the epithalamic pineal organ, a gland involved in a wide range of physiological functions. The aim of this study was to investigate whether the stress effects on the pineal gland are related with changes in the expression of neurotrophic factors and cell adhesion molecules. Using reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, we analyzed the effect of chronic immobilization stress on the BDNF mRNA and integrin alpha5 expression in the rat pineal gland. We found that BDNF is produced in situ in the pineal gland. Chronic immobilization stress induced upregulation of BDNF mRNA and integrin alpha5 expression in the rat pineal gland but did not produce changes in beta-actin mRNA or in GAPDH expression. Stressed animals also evidenced an increase in anxiety-like behavior and acute gastric lesions. These results suggest that BDNF and integrin alpha5 may have a counteracting effect to the deleterious actions of immobilization stress on functionally stimulated pinealocytes. Furthermore, this study proposes that the pineal gland may be a target of glucocorticoid damage during stress.

Pituitary adenylate-cyclase activating polypeptide (PACAP) is a neuropeptide originally isolated from the hypothalamus and located in many neuronal systems in both the central and peripheral nervous system. PACAP is also found in nerve fibers innervating the pineal gland, where it stimulates the secretion of the pineal hormone, melatonin, by binding to specific PACAP-receptors located on the cell membrane of the pinealocyte. In this study we have investigated the origin of PACAP-containing nerve fibers innervating the rat pineal gland by combined retrograde tracing with Fluorogold and immunohistochemistry for PACAP. A solution of 2% Fluorogold was injected iontophoretically into the superficial pineal gland of Wistar rats, and the animals were allowed to survive for 1 week. After perfusion fixation of the rats, the location of the tracer was investigated in the brain and the sphenopalatine, otic, and trigeminal ganglia. The tracer was found in all the investigated ganglia. However, colocalization with PACAP was predominantly found in the trigeminal ganglion and only occasionally in the sphenopalatine and otic ganglia. Due to the stimulatory function of PACAP on pineal melatonin secretion, the PACAP-containing neurons of this ganglion could be considered a subset of the parasympathetic nervous system. The presence of neurons with a parasympathetic function in a ganglion that has been considered a purely sensory ganglion, is a new concept in neuroanatomy.

Physicians have noted since antiquity that their patients complained of less pain and required fewer analgesics at night-time. In humans, the circulating levels of melatonin, a pineal substance with analgesic and hypnotic properties, exhibit a pronounced circadian rhythm with serum levels being high at night and low during day-time. Moreover, pinealectomy abolishes the analgesic effects of melatonin, and naloxone disrupts the day-night rhythm of nociception. In this study, we have attempted to identify and characterize the nature and types of opioid receptor in bovine pinealocyte membranes, using a radioligand binding technique with the selective radioligands [3H]DAMGO, [3H]DPDPE, [3H]U69593 and [3H]orphanin-FQ (OFQ) for identifying mu (mu)-, delta (delta)-, kappa (kappa)- and opioid receptor-like (ORL(1)) receptors, respectively. The saturation experiments on bovine pinealocyte membranes for [3H]DPDPE binding provided B(max) and K(d) values of 553+/-24 fmol/mg protein and 1.3+/-0.6 nM; and for [3H]DAMGO binding provided B(max) and K(d) values of 6.3+/-1.3 fmol/mg protein and 1.2+/-0.4 nM, respectively. On the other hand, the specific radioligands ([3H]U69593 and [3H]OFQ) binding of kappa and ORL(1) receptors were undetectable in bovine pinealocyte membranes. Furthermore, competitive experiments with opioid agonist and antagonist and related compounds confirmed the presence of mu- and delta-opioid binding sites in bovine pinealocyte membranes. These results indicate that neither kappa nor ORL(1) receptors are present on the pinealocytes, and the majority of opioid receptors found in the bovine pineal gland are delta (possibly, both delta(1) and delta(2)) types, with a minority being mu type, and that both are primarily located on the bovine pinealocyte membranes. These opioid receptors, by stimulating the activity of N-acetyltransferase, enhance the synthesis of melatonin.

The mammalian pineal gland is known to receive a noradrenergic sympathetic efferent signal from the suprachiasmatic nucleus (SCN) via the superior cervical ganglion. Arg-vasopressin (AVP) containing neurons in the SCN is one of the output paths of circadian information to the other brain areas. AVP release from the SCN is suppressed by melatonin. In turn, we determined the direct effect of AVP on melatonin release using pineal gland explant culture. AVP (1 microM) suppressed melatonin release. Noradrenaline stimulated melatonin release was attenuated by AVP. In turn, the expression of the melatonin synthesis enzyme arylalkylamine N-acetyltransferase mRNA in the rat SCN was reported. We measured melatonin content in the SCN in rats kept under the light-dark cycle and constant dim light. Melatonin in the SCN was higher during the dark period than that in the light. A similar tendency was also observed in the SCN of animals kept under a constant dim light. It was suggested that the reciprocal regulation of melatonin release and AVP release occurs in the SCN and pineal gland.

The chicken pineal gland, which contains a heterogeneous cell population, sustains a circadian rhythm of activity. Synchronization of cellular activity of heterogeneous cells might be facilitated by gap junctional intercellular channels which are permeable to ions and second messengers. To test this possibility, we looked for morphologically identifiable gap junctions between the different pineal cells, used antibodies and cDNA probes to screen for the presence of connexins, and tested for functional intercellular coupling. By transmission electron microscopy and immunocytochemistry, gap junctions and connexins were observed between pinealocyte cell bodies, stromal cells, astrocytes, and astrocyte and pinealocyte processes. Two gap junctional proteins, connexin43 and connexin45, were detected by immunocytochemistry, immunoblotting and RNA blot analysis. Functional intercellular coupling was observed in the gland by transfer of low molecular weight dyes. Dye transferred between homologous and heterologous cells. These data suggest that homologous and heterologous gap junctions may provide a mechanism for coordination of the cellular responses of the elements of the biological clock which are induced by lighting cues to produce the circadian rhythm of pineal activity.

Conventional and immunocytochemical, light- and electron-microscopic studies on the innervation of the pineal gland of the tree shrew (Tupaia glis) were made. Neuropeptide Y (NPY)-immunoreactive fibers, which were abundantly distributed in the gland, disappeared almost completely after superior cervical ganglionectomy, suggesting that these fibers are mostly postganglionic sympathetic fibers. By contrast, tyrosine hydroxylase (TH)-immunoreactive fibers, which were less numerous than NPY-fibers, remained in considerable numbers in ganglionectomized animals, indicating the innervation of TH-positive fibers from extrasympathetic sources. Bundles of substance P (SP)- or calcitonin gene-related peptide (CGRP)-immunoreactive fibers, entering the gland at its distal end, were left intact after ganglionectomy. SP-fibers were numerous, but CGRP-fibers were scarce in the gland. SP-immunoreactive fibers were myelinated and nonmyelinated, and were regarded as peripheral fibers because of the presence of a Schwann cell sheath. NPY- and SP-immunoreactive fibers and endings were mainly localized in the pineal parenchyma. NPY-immunoreactive endings synapsed frequently, and SP-positive ones did less frequently, with the cell bodies of pinealocytes. The results suggest that NPY and SP directly control the activity of pinealocytes. Sections stained for myelin showed that thick and less thick bundles of myelinated fibers entered the gland by way of the habenular and posterior commissures, respectively. Under the electron microscope, the bundles were found to contain also unmyelinated fibers. A considerable number of nerve endings synapsing with the cell bodies of pinealocytes remained in ganglionectomized animals; these endings were not immunoreactive for TH or SP. Such synaptic endings may be the terminals of commissural fibers.

Recent studies have strengthened the hypothesis that neuroactive amino acids such as L-glutamate play an important role in the physiology of the mammalian pineal gland. In particular, there is now considerable evidence that L-glutamate is liberated from electron-lucent microvesicles of pinealocytes for a paracrine modulation of melatonin synthesis and release which may at least partially be mediated by the metabotropic glutamate receptor mGluR3. In order to expand our incomplete knowledge of possible pineal target cells and signal transduction mechanisms which are involved in glutamate-dependent intercellular communication, we have performed an immunohistochemical study of the gerbil pineal gland with antibodies directed against the metabotropic glutamate receptors mGluR2/3 and mGluR5. Using microwave irradiation of cryostat sections prior to immunostaining, strong immunoreactivity for both receptor subtypes was constantly observed in a subpopulation of pineal cells. Interestingly, these mGluR-positive cells could be identified as interstitial glial cells since they were labeled by antibodies against the intermediate filament protein vimentin in double immunofluorescence histochemistry. This indicates that interstitial glial cells in the gerbil possess the capacity to express at least two metabotropic glutamate receptors coupled to different intracellular signal transduction pathways. Therefore, it can be concluded that the glutamatergic communication system of the pineal gland may not only enable paracrine crosstalk among pinealocytes but probably also relies on interactions between pinealocytes and interstitial cells analogous to neuronal-glial signaling.

Chick pineal cells express a circadian rhythm of melatonin release under light–dark (LD) cycles, with an increase during the dark period and a decrease during the light period, and this rhythm persists under constant darkness (DD). We cultured individual single pineal cells with 15 μl of medium per well in a Terasaki plate and measured melatonin secretion every 12 h under LD, DL and DD. Individual cells secreted more melatonin during the dark period than during the light period under both LD and DL conditions, and those rhythmic secretions persisted under DD. These results suggest that individual pineal cells in chick have photoreceptive, circadian clock and melatonin-synthesizing capacities.

By means of the fura-2 technique and image analysis the intracellular concentration of free calcium ions [Ca 2+ ] i was examined in isolated rainbow trout pinealocytes identified by S-antigen immunocytochemistry. Approximately 30% of the pinealocytes exhibited spontaneous [Ca 2+ ] i oscillations whose frequency differed from cell to cell. Neither illumination with bright light nor dark adaptation of the cells had an apparent effect on the oscillations. Removal of extracellular Ca 2+ or application of 10 μ M nifedipine caused a reversible breakdown of the [Ca 2+ ] i oscillations. Application of 60 mM KCl elevated [Ca 2+ ] i in 90% of the oscillating and 50% of the non-oscillating pinealocytes. The effect of KCl was blocked by 50 μ M nifedipine. These results suggest that voltage-gated L-type calcium channels play a major role in the regulation of [Ca 2+ ] i in trout pinealocytes. Experiments with thapsigargin (2 μ M) revealed the presence of intracellular calcium stores in 80% of the trout pinealocytes, but their role for regulation of [Ca 2+ ] i remains elusive. Treatment with norepinephrine (100 pM–50 μ M), previously shown to induce calcium release from intracellular calcium stores in rat pinealocytes, had no apparent effect on [Ca 2+ ] i in any trout pinealocyte. This finding conforms to the concept that noradrenergic mechanisms are not involved in signal transduction in the directly light-sensitive pineal organ of anamniotic vertebrates.

We karyometrically investigated the nucleus suprachiasmaticus (SCN) which had been manipulated in several ways in order to analyze the functional importance of the pineal gland on the primary pacemaker of mammals in the course of the year. The manipulation modes were (i) pinealectomy (PX), and (ii) sympathetic denervation of the pineal by bilateral extirpation of the upper cervical ganglia (ganglionectomy, GX). Additionally, the influence of the inactivated pineal, obtained through hypothyroidism which was realized by (iii) subtotal thyroidectomy (TX), was also investigated. With respect to annual oscillations the results of our investigations were able to illustrate the following. (1) The SCN consists of at least two parts (ventrolateral and dorsomedial) each with different functions and relationships. The nuclei of the ventrolateral cells are bigger and there are many indications both in our own research and from literature that the neurons of this part are involved in the generation of rhythms. (2) The size of the cell nuclei of the ventrolateral part shows annual patterns. In the course of the year the maxima of the nuclear volume were registered in March and September (bimodal pattern, equinox, L:D = 12:12). PX, GX or TX only negligibly changed the bimodal annual pattern. However, in comparison the smallest cell nuclei were registered in the winter (short day). The much smaller cell nuclei of the dorsomedial part likewise show bimodal patterns but only in the experimental groups. The control group of this part shows an unimodal annual curve with a minimum at long-day conditions (June, L:D = 16:8). (3) All manipulations which inactivated the pineal or reduced the content of melatonin (PX, GX, and TX) were followed by an increase (activation) of cell nuclei of the SCN. In contrast to these effects, an increase of thyroxine (by exposure to cold), has an opposite effect (not documented here). In conclusion these results indicate, without a doubt, that a negative correlation exists, functionally, between the SCN and the pineal (in the same annual experiment the nuclear size of the pinealocytes was increased, under short-day conditions in December, and decreased under long-day circumstances in June). Additionally, it could be shown that the degree of negative correlation between the pineal and the SCN was seasonally dependent. The lowest effects of PX, GX and TX were registered at short-day conditions (December, L:D = 8:16).

There is ample evidence that beta-adrenergic stimulation of cyclic GMP formation is potentiated by alpha1-adrenergic mechanisms, the latter leading to elevation of intracellular Ca2+ concentration ([Ca2+]i) and protein kinase C (PKC) activation. Recent studies have shown that nitric oxide synthase (NOS) and nitric oxide (NO) are a component of the adrenoceptor-cyclic GMP signalling pathway. The aim of the present investigation was to study the roles of alpha1-adrenergic mechanisms, Ca2+ and PKC on NO-stimulated cyclic GMP formation. To this end suspension cultures of rat pinealocytes were treated with the NO donor sodium nitroprusside (SNP) in the presence of alpha1-adrenergic agonists, [Ca2+]i-elevating substances, PKC inhibitors, followed by measurement of cyclic GMP accumulation. It was found that alpha1-adrenergic stimulation did not affect NO-activated cyclic GMP synthesis. Therefore alpha1-mechanisms act prior to NO induction of cyclic GMP. Agents, which elevate [Ca2+]i depressed NO-induced cyclic GMP formation. Since literature data show that Ca2+ stimulates pineal NO formation it is apparent that Ca2+ has antagonistic effects in the pineal adrenoceptor-cyclic GMP signalling pathway. The inhibitory effect of Ca2+ was unchanged after inhibition of phosphodiesterases suggesting that it may interfere with cytosolic guanylyl cyclase activation. Inhibition of PKC, but not of other protein kinases, decreased NO-activated cyclic GMP formation. Therefore it appears that non-alpha1-adrenergic-regulated PKC possesses a regulatory rote in NO-induced cyclic GMP formation.

Recent evidence points the secretion of serotonin from the rat pineal gland both in vivo and in vitro. In view of the fact that adrenergic stimulation of cultured pinealocytes leads to rapid serotonin release well in advance of melatonin production, the question arises as to the physiological significance of serotonin secretion. The present studies examined the impact of ketanserine, a specific serotonin type 2 receptor antagonist, on the cyclic AMP and melatonin response of cultured rat pinealocytes to adrenergic stimuli. Whereas the β-adrenergic agonist isoproterenol (1 μM) stimulated cAMP accumulation as well as melatonin release significantly, the presence of ketanserine inhibited these responses in a dose-dependent manner. Since the same inhibitory effect of ketanserine was seen in cells stimulated by dibutyryl-cAMP (which acts post-adrenergic receptor to elevate melatonin synthesis), the mechanism for serotonin's feedback actions does not appear to involve changes in adrenergic receptor/cAMP coupling. These results indicate that extracellular serotonin may be important for the full activation of melatonin secretion following adrenergic stimulation.

Melatonin, an indoleamine hormone synthesized in the pinealocytes, has been implicated as influencing the intake of alcohol in rats. It has been shown that this hormone is voltammetrically electroactive at the surface of pretreated carbon fiber microelectrodes in vitro and in vivo, in rat cerebral melatonergic regions such the pineal gland. The aim of this work consisted in the study of the influence of melatonin on spontaneously ethanol drinking or ethanol avoiding rats selected throughout a free choice two bottle test. It appeared that only the water preferring rats were affected by treatment with the hormone and that in vivo voltammetric related levels of melatonin were higher in the pineal gland of ethanol drinking rats versus water preferring rats. In addition, when treated with the melatonin antagonist GR128107 ethanol drinking rats significantly reduced the spontaneous consumption of alcohol.

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 TM , 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 increase [Ca 2+ ] i . This development makes it possible to conduct routine single-cell image analysis and other studies of freshly isolated rat pinealocytes.

Nerve growth factor receptor immunoreactivity (NGFRI) in the pineal gland was examined both light and electron microspically using the monoclonal antibody 192 IgG. NGFRI was located on sympathetic fibers and on perivascular cells resembling macrophage/microglia. A pineal gland dispersed cell culture model confirmed the presence of NGFRI in cellsthat exhibited processes of varying lengths and were distributed among pinealocytes and other cells. Pinealocytes in dispersed cell culture were identified immunocytochemically by their expression of S-antigen, their round shape and small size and their tendency to extent neurites in the direction of the flat cells in culture. The length of pinealocyte neurites showed a significant increase when cultured in the presence of NGF (25 ng/ml), suggesting that trophic factors, mediated by these macrophage/microglial cells, are important to the morphogenesis of these neuroendocrine cells. Neutotrophic activation of these neuroendocrine macrophage/microglia may have neuro-immunomodulatory implications leading to expression of proteins encoded by the major histocompatibility complex.

The effect of pituitary adenylate cyclase polypeptide (PACAP) on rat pineal was examined. PACAP stimulated melatonin release from cultured dissociated pinealocytes with a 10(4) higher potency than isoproterenol (EC50 were 30 pM and 250 nM, respectively). The 10(-9) M PACAP stimulation was not inhibited by 5 x 10(-6) M VIP antagonist whereas that of 10(-9) M VIP was reduced by 54%. Kinetic analysis of melatonin release indicated that PACAP acts postsynaptically via receptor activation.

The monoclonal antibody WF6, raised against purified Torpedo nicotinic acetylcholine receptor (nAChR) was used to study the distribution of cholinoceptors in the rat pineal gland by means of Western blot analysis, light- and electron microscopy. The immunoblot analysis using homogenized pineal gland revealed a labeled protein band of apparent molecular weight 40 kDa which was identified as α-subunits of a nAChR. In the light microscope, approximately one-fourth of the pinealocytes exhibited cytoplasmic immunoreactivity (IR) of varying density. In the electron microscope, IR was seen as patchy staining of cell membranes of pinealocyte somata and processes. Presynaptic IR material was not found. Distribution and intensity of the observed IR was not significantly different in pineal sections from ganglionectomized rats, nor were any alterations found that would relate to the animals' sex or to the time of killing (day vs night). Our results provide further evidence for the existence of cholinergic receptors in the mammalian pineal. They may be important for the understanding of the gland's regulation.

An immunohistochemical investigation of the bovine pineal gland was performed using both a rabbit polyclonal antibody and a rat monoclonal antibody against choline acetyltransferase (ChAT). A network of ChAT-immunoreactive (IR) nerve fibers was located throughout the pineal gland, both in the perivascular spaces and between the pinealocytes. Most of the intrapineal ChAT-IR nerve fibers were endowed with varicosities. In addition, some ChAT-IR intrapineal neurons were found, often located at the base of the gland near the pineal recess. Within the habenular nucleus and pineal stalk, ChAT-IR perikarya and nerve fibers were also present. Some of these fibers projected towards the pineal gland. A number of ChAT-IR nerves were also located in the posterior commissure and could be followed into the gland. At the caudal tip of the pineal gland, a bundle of ChAT-IR nerve fibers was observed to penetrate into the gland together with blood vessels. The presence of a cholinergic innervation of the bovine pineal gland, together with previous demonstration of the presence of choline acetyltransferase and muscarinic receptor binding sites in the bovine pineal gland, indicates a functional influence of a cholinergic nervous system on the pinealocyte.

Melatonin is synthesized by the vertebrate pineal gland in a circadian fashion and is involved in numerous circadian and seasonal processes in the organism. The vertebrate retina also produces melatonin rhythmically to regulate rhythmic physiological processes in the eye. In both organs, melatonin is synthesized from serotonin by the sequential action of the enzymes, N-acethyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT), and can be stimulated by increases in cyclic AMP through a mechanism requiring protein synthesis. The regulation of ocular melatonin biosynthesis in mammals and particularly humans, has not been well studied. Recently, we have shown that Y79 human retinoblastoma cells produce melatonin and that cAMP can stimulate melatonin production. Y79 cells, therefore, provide a model system in which to study melatonin synthesis in human tissue. We report that cAMP stimulates NAT, but not HIOMT activity in Y79 cells, and that stimulation of NAT activity is linearly related to melatonin release. In addition, the stimulation of NAT and melatonin requires protein synthesis. The turnover of NAT is rather rapid, with a half-life of about 20 min. These results suggest that the regulation of melatonin in Y79 retinoblastoma cells is similar to that found in the retina and pineal of other vertebrates.

The duration of the nocturnal interval during which light can induce high Fos-like immunoreactivity in the suprachiasmatic nucleus is a function of the photoperiod to which rats are exposed. The effect of photoperiod is not altered by pinealectomy indicating that day length affects the functional state of the suprachiasmatic nucleus circadian pacemaking system in the rat directly, not via the pineal melatonin signal.

The effects of sleep deprivation with or without melatonin treatment on the pineal morphology in rats were studied. Five days after sleep deprivation and using electron microscopy, many of the pinealocytes exhibited structural alterations including dilation of the cisternae of the rough/smooth endoplasmic reticulum, Golgi saccules and mitochondria, and an increase in the numbers of lipid droplets, vacuoles and dense-core vesicles. These features were considered as morphological evidence of increased synthesis or secretion by the pineal gland. In addition, numerous membranous profiles, considered to be degraded cellular organelles, were observed in some pinealocytes and sympathetic nerve terminals. It is suggested that the occurrence of degenerating organelles had resulted from the deleterious effect of sleep deprivation. This may be attributed to an overload of secretory activity of the pineal gland during stress elicited by the long-term sleep deprivation, leading to functional exhaustion and irreversible damage of the oxidation-related organelles. In sleep-deprived rats receiving a single injection of melatonin (10 mg/kg) for 5 consecutive days, the above features indicative of pinealocytic activation were attenuated. In fact, all signs of degeneration of cellular organelles were rarely found. These results suggest that the pineal gland is itself a target for exogenously administered melatonin. Thus, melatonin when administered systemically may be used as a potential neuroprotective drug against neuronal damage induced by sleep deprivation.

To investigate the functional involvement of the pineal gland in circadian expression of the rat period homolog gene (rPer2) in the suprachiasmatic nucleus (SCN) and peripheral tissues, we performed Northern blot analysis in tissues from pinealectomized rats. The ectomy did not have any significant effects on rPer2 mRNA expression patterns both in a daily light-dark condition and in a constant darkness. These results suggest that the rhythmic secretion of pineal melatonin is not essential for the circadian expression of clock genes in the SCN and other peripheral tissues of rats.

Rat pinealocytes use L-glutamate as a modulator for melatonin synthesis. Upon binding of L-glutamate to the class II metabotropic glutamate receptor, norepinephrine (NE)-dependent formation of cAMP was inhibited, resulting in decreased serotonin-N-acetyltransferase (NAT) activity and melatonin output. Although L-glutamate at 1 mM caused 90% inhibition of melatonin synthesis, about 30% of the NAT activity remained, suggesting the presence of another target for L-glutamate. In this study, we found that L-glutamate also inhibits hydroxyindole-O-methyltransferase (HIOMT). The inhibition is reversible and dose-dependent: the maximal inhibition was obtained with more than 0.4 mM L-glutamate. Contrary to L-glutamate-evoked inhibition of NAT, agonists for class II metabotropic receptors such as (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG IV) had no effect on HIOMT. Neither (2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)glycine (MCCG), an specific antagonist for class II mGluRs, nor dibutyryl cAMP restored the L-glutamate-evoked inhibition of HIOMT. Northern blot analyses revealed that L-glutamate significantly inhibits the expression of mRNA of NAT, but not that of HIOMT. These results indicated that HIOMT is an another target for L-glutamate due to its inhibition of melatonin synthesis, and the signaling pathway toward the inhibition is distinct from that of NAT.

In the rat pineal gland, norepinephrine activates α1- and β-adrenergic receptors and triggers melatonin production through an increase in the intracellular calcium concentration ([Ca2+]i) and stimulation of the cAMP/cAMP responsive element-binding protein (CREB) cascade. VIP and PACAP also elevate the intracellular cAMP level and promote melatonin formation. Finally, ACh antagonizes the norepinephrine-induced hormone synthesis via nicotinic acetylcholine receptors and subsequent activation of voltage-gated calcium channels. By immuno(cyto)chemical demonstration of phosphorylated CREB and calcium imaging we have investigated the temporal relationship between the maturation of these signaling pathways and the rhythmic onset of melatonin biosynthesis in developing rat pinealocytes. Norepinephrine-regulated calcium signaling and phosphorylation of CREB are already fully developed at birth, i.e., prior to ingrowth of the sympathetic innervation into the pineal parenchyma, and appear to develop in an innervation-independent manner. VIP- and PACAP-induced CREB phosphorylation is restricted to subpopulations of neonatal cells and thus also displays an adult pattern. Cholinergic calcium signaling exhibits a developmental switch within the first three postnatal weeks. In neonatal pinealocytes, acetylcholine elevates [Ca2+]i via muscarinic rather than nicotinic acetylcholine receptors. In the second postnatal week, pinealocytes gain responsiveness to nicotine and gradually lose responsiveness to muscarinic cholinergic stimuli. Voltage-gated calcium channels are absent in neonatal cells and develop during the first postnatal days. ACh-evoked cellular events may be diversified depending on the functional subclass of receptor that is present. The transient existence of muscarinic acetylcholine receptors and the subsequent switch to nicotinic receptors would permit ACh to elicit temporary effects in early pineal development.

Genetically mutant anophthalmic rats lacking a complete visual system due to the absence of eyeballs and optic nerves up to the optic chiasma were used as a model to study photo-regulated physiological activities. The photoreception in these mutant rats was determined by measuring the neuroendocrine response of the pineal gland-melatonin levels in the serum, and synaptic ribbon complexes (SRCs) in the pinealocytes. These parameters were studied in both normal and anophthalmic rats maintained under light-dark (LD 12:12), continuous dark (DD) and light (LL) conditions. Both normal and mutant anophthalmic animals showed nocturnal increases in serum melatonin levels and in the number and diameter of SRC and their vesicles in the pinealocytes in LD. The daily rhythms persisted even upon transfer to DD both in normal and mutant rats, whereas in LL, the nocturnal elevation of both the parameters disappeared. These observations suggested that congenitally blind rats can perceive light. The studies of these parameters in both normal and mutant rats in reversed-LD conditions confirmed that pineal rhythms can be entrained by light-dark cycles in congenitally anophthalmic mutant rats through a nonvisual system for light perception.

Hydroxyindole-O-methyltransferase (HIOMT) catalyses the last step of all the 5-methoxyindoles synthesized in the pineal gland. The synthetic activity of this neuroendocrine structure is driven not only by noradrenaline but also by various neuropeptides. Recently we have established (1) that one of these neuropeptides, neuropeptide Y (NPY), stimulates specifically HIOMT activity in rat pinealocytes and (2) that the density of the NPY-immunoreactive (NPY-IR) fibers innervating the pineal gland of the European hamster (Cricetus cricetus) displays seasonal variations with a large increase in the late autumn. These findings have led us to evaluate a possible seasonal control of NPY on the European hamster pineal gland. We thus compared the nycthemeral patterns of pineal HIOMT activity and 5-methoxytryptophol (5-ML) content and of circulating MEL levels in European hamsters when NPYergic innervation is low (end of October) and when it is the highest (mid-December). We report in this study that HIOMT activity is significantly increased by 80% in mid-December compared with end of October. This increase is correlated with the appearance of a nycthemeral rhythm of pineal 5-ML levels (with a fourfold increase occurring in early dawn and decreasing slowly towards the end of the day). These observations suggest that NPY could be an important neurotransmitter involved in the seasonal control of the biochemistry of the European hamster pineal gland via a stimulatory effect on HIOMT activity.

In the rat pineal gland the glycogen stores were cytochemically localized in astrocytes and pinealocytes. Moreover, it was found that norepinephrine (NE) induced a time- and concentration-dependent reduction in pineal glycogen content and yielded lactic acid. The NE effect was prevented by blocking alpha1- but not alpha2 or beta-adrenoceptors. Activation of alpha2-adrenoceptors induced a small decrease in glycogen levels that could have pre- and postsynaptic components. Activation of beta-adrenoceptors with 10(-12)-10(-3) M isoproterenol (ISO) induced a bell shape concentration-response curve, presumably due to desensitization, since the response induced by 10(-4) M ISO was greater with shorter period of stimulation. On the other hand, activation of alpha1-adrenoceptors with 10(-12)-10(-3) M phenylephrine (PHN) induced a hyperbolic concentration-response curve with a maximum at concentrations above 10(-8) M. Moreover, treatment with ISO drastically reduced the response induced by PHN concentrations lower but not higher than 10(-6) M, favoring a concentration-dependent response between 10(-6) and 10(-4) M PHN, similar to that induced by equimolar NE concentrations. Thus, the NE-induced reduction in glycogen content of the rat pineal gland is mainly mediated by alpha1-adrenoceptors and modulated by intracellular mechanisms activated by beta-adrenoceptors.

Melatonin release was measured from zebrafish pineal organs and retinas maintained in flow-through culture. Pineal organs released melatonin in a strong circadian rhythm through 5 days in constant darkness, and the phase of this rhythm was reset by in vitro exposure to phase-shifted light cycles. In contrast, the retinal melatonin rhythm rapidly damped out in constant darkness, even in the presence of (phase-shifted) light cycles. The zebrafish pineal should be useful for in vitro studies of vertebrate circadian clock mechanisms.

Melatonin release in a pineal cell culture from 13- and 14-day-old chick embryos increased during the dark phase and decreased during the light phase of a 12 h light:12 h dark cycle. When the light-dark cycle was reversed, the pattern of melatonin release in the culture also reversed. 8-Bromo cyclic-AMP stimulated melatonin release in both the light and dark phases. However, no rhythm of melatonin release was detected under constant dark (DD) conditions in a cell culture from 14-day-old chick embryos. In 18-day-old chick embryos, the pineal cell culture expressed a circadian rhythm of melatonin release under DD conditions. These results indicate that mechanisms regulating melatonin synthesis in the avian pineal gland are established during embryonic life.

We compared the circadian oscillation of melatonin release from cultured pineal cells in Japanese quail, pigeon and house sparrow, to determine whether the pineal gland of these species retains the circadian oscillator function in vitro. After dissociated pineal cells have been cultured for 4 days under 12 h : 12 h light-dark (LD) cycle, they were perfused at a flow rate of 0.25 ml/h for 6–7 days under LD or constant darkness (DD). Melatonin release increased during the dark period and low during the light period in all pineal cell cultures. Under DD conditions, the circadian rhythm of melatonin release persisted for up to 3–4 cycles in pigeon and house sparrow pineal cells, but the amplitude of the rhythm decreased gradually. However, in Japanese quail pineal cell culture the circadian oscillation of melatonin release was weak or abolished under DD conditions. These results strengthen the argument that the avian pineal gland;s role in circadian organization differs between species. The direct demonstration of species-specific differences in the mechanism of the circadian oscillation of melatonin release from pineal cells should provide a useful model for the analysis of the pineal's circadian system at the cellular level.

Recoverin is a recently discovered 26 kDa calcium-binding protein, which activates guanylate cyclase in retinal photoreceptors when the intracellular concentration of free calcium drops upon photoexcitation. In this study we examined the distribution of recoverin in retinae and pineal organs of Xenopus laevis larvae, 1-day-old chicken, adult pigeon, albino rat, sheep and man by means of immunocytochemistry. Recoverin immunoreaction was found in all species investigated except for the chicken. In the retina, recoverin immunoreaction was restricted to photoreceptors; all other cell types were immunonegative. In the pineal organ, the recoverin immunoreaction labeled ‘pinealocytes of the sensory line’, i.e. classical pineal photoreceptors of Xenopus laevis larvae, modified pineal photoreceptors of pigeon, and pinealocytes of mammals. The number of recoverin immunoreactive pinealocytes varied considerably among species of mammals; very few cells were stained in the rat pineal organ, whereas in rabbit, sheep and man, numerous pinealocytes were found to be recoverin-immunoreactive. No immunocytochemical staining was observed after preabsorption of the recoverin antibody with the recombinant protein. Immunoblotting experiments showed that the immunoreaction is due to a protein of 26 kDa in both retina and pineal tissue. Thus, recoverin appears to belong to the family of proteins which are expressed in both retina and pineal organ and are highly conserved in the course of phylogeny. Recoverin may be involved in phototransduction in the directly light-sensitive pineal organs of poikilothermic vertebrates and birds. However, the functional role of recoverin in the mammalian pineal organ, which is not photosensitive, remains unknown.

The pineal hormone melatonin may regulate seasonal reproduction and entrainment of circadian rhythms by binding to specific brain receptors. An analysis of melatonin receptor distribution in the lizard brain revealed an asymmetry of melatonin binding in the diencephalon. A high degree of melatonin binding was present in the left habenular nucleus, but no binding was observed in the habenulum of the right brain hemisphere. It is intriguing that the left habenular nucleus, in contrast to the right habenulum, both possesses a high density of melatonin receptors and receives primary photic input from the parietal eye. Similarly, the optic tectum, which receives primary visual input from the retina, is also rich in melatonin receptors. These observations suggest that the left habenulum is under dual control (neuronal and hormonal) of the parietal eye/pineal complex, and that melatonin may play a significant role in neural processing of visual information.

Pinealocytes and retinal photoreceptor cells contain an unusual cytoplasmic complex composed of the G beta gamma dimer of GTP-binding regulatory proteins (G-proteins) tightly bound to an acidic 33 kDa phosphoprotein termed MEKA or phosducin; MEKA is a substrate of cyclic AMP-dependent protein kinase. This study characterized the developmental appearance of these and two related proteins, G gamma and S-antigen, in pineal and retinal tissue. MEKA was absent in the pineal gland prior to birth, at a time when it was possible to detect G beta in pineal cytoplasm, indicating that the appearance of G beta in the cytoplasm precedes that of MEKA and does not appear to require the presence of MEKA. The absence of MEKA at this time indicates that the cyclic AMP stimulation of pineal serotonin N-acetyltransferase activity is not mediated by MEKA, which has been considered as a possible role of MEKA. After postnatal day 7, pineal MEKA and cytoplasmic G beta increased in a parallel manner, with peak values occurring at about postnatal day 21. Thereafter, both proteins in the pineal gland decreased in a parallel fashion to 10 and 35% of their peak values, respectively; in contrast, the cytoplasmic protein S-antigen and membrane associated G beta remained at maximal levels after this time. Whereas both MEKA and G beta decreased late in development in the pineal gland, these proteins either increased or remained constant in the retina. These tissue-specific patterns were found to differ from those of another cytosolic protein found exclusively in the pineal gland and retina, S-antigen, which remained constant after day 21 in the pineal gland but decreased in the retina late in life.

Dye coupling was observed between pinealocytes in acutely dissected pineal glands of adult rats. Pinealocytes maintained in culture were also electrically coupled. Connexins 26 and 43 and their respective mRNAs were present but neither connexin32 nor itsmRNA were detected. Pinealocytes expressed only connexin26 whereas connexin43 was confined to astrocytes. In 5-day-old cultures of pinealocytes the incidence of dye coupling and level of immunodetectable connexin26 were low, and both were increased by norepinephrine (NE). The increase in incidence of coupling was maximal at around 6 h after treatment and was prevented by inhibitors of protein of mRNA synthesis. NE-induced metabolic and electrical synchronization mediated by gap junctions may favor melatonin secretion.

Environmental lighting can prevent or quickly reverse the nocturnal elevation of serotonin N-acetyltransferase activity in the rat pineal gland. Intraventricular injections of carbachol, acting via a 'nicotonic' cholinergic receptor, mimic the acute effect of light. Injections of alpha-bungarotoxin near the suprachiasmatic nucleus of the hypothalamus prevent the effects of light. These results suggest that acetylcholine is involved in the effects of light on the rat pineal's circadian rhythm.

The effect of suprachiasmatic nucleus (SCN) ablation on rhythms in brain neurotransmitter receptors was studied. In control animals, the rhythms in the number of α-adrenergic and benzodiazepine receptors were circadian, whereas the rhythms in s-adrenergic and acetylcholine receptors were ultradian. SCN ablation resulted in loss of circadian, but not ultradian, rhythms, without change in the 24-h mean numbers. SCN ablation appears to cause a selective loss of circadian function in the regulation of brain receptors.

The uptake of the inhibitory transmitter substance gamma-aminobutyric acid (GABA) into the adult rat pineal gland was studied autoradiographically using both light and electron microscopy. The sites of GABA uptake were shown to be exclusively present in the gliocyte cells of the gland following both in vitro incubation with tritiated GABA and after in vivo administration of the amino acid by intra-arterial injection. Both the pinealocyte cells and the numerous sympathetic axons in the gland were devoid of silver grains. Preliminary biochemical studies indicated that the gliocyte uptake process for GABA resembles that in the satellite glia of the sensory ganglia but differed from that in slices of the cerebral cortex. Evidence is also presented which shows the pineal gland to contain endogenous GABA and the enzymes directly associated with its in vivo metabolism, L-glutamate-1-carboxylase (EC 4.1.1.15) (GAD) and GABA-2-oxoglutarate aminotransferase (EC 2.6.1.19) (GABA-T). Furthermore, a 3-fold rise in endogenous GABA occurred in the pineal after inhibition of GABA-catabolism as would be expected if the GABA-shunt pathway was functionally active in the oxidative metabolism of the pineal gland.

Using the fluorescence histochemical technique, yellow autofluorescent granules were observed in neurones of the arcuate and ventromedial hypothalamic nuclei of the rat (type I cells). In the same nuclei, neurones could be demonstrated showing a formaldehyde-induced yellow fluorescence (type II cells). Microelectrophoresis and special staining methods applied to the pineal gland revealed the autofluorescent compound to be a protein containing a relatively high content of tryptophan. It is probable that the formaldehyde-induced yellow fluorescence is due to the presence of serotonin. In view of investigating a possible functional relationship between the pineal gland and the parvocellular hypothalamic nuclei mentioned, hypothalamic type I and type II cells, as well as autofluorescent and serotonin-containing pinealocytes, if present, were quantified under the following experimental conditions: (1) p-chlorophenylalanine (pCPA) administration, (2) castration, (3) pinealectomy, and (4) pinealectomy followed by substitution using rat and sheep pineal extract. Administration of pCPA caused a decrease in the number of type II and an increase in the number of type I cells, both in the pineal gland and the hypothalamic nuclei. Castration, in contrast, was followed by an increase in the number of autofluorescent pinealocytes, but a decrease of autofluorescent neurones in the hypothalamic nuclei (type I cells) while the number of serotonin-containing pinealocytes increased; decreasing in both hypothalamic nuclei. After pinealectomy the hypothalamic nuclei showed an increase of type I neurones, but a decrease of type II nerve cells. Pinealectomy followed by substitution using pineal extracts restored the number of type I and type II neurones to that normally found in the arcuate and ventromedial hypothalamic nuclei of control animals. The present investigation brings histological evidence of an influence exerted by the rat pineal gland on nuclei forming part of the hypothalamic hypophyseotropic area. The data obtained and some of the literature strongly suggest that the type II neurones, which probably contain serotonin, inhibit, in the same hypothalamic nuclei, the production of luteinizing hormone-releasing factor (LH-RF). As yet, the function of the autofluorescent compound present in the type I neurones is not known.

The presence of the glial marker proteins, the S-100 and the glial fibrillary acidic (GFA) protein, in the pineal gland was investigated in the rat. Using both the indirect peroxidase-labelled immunoglobulin technique and the unlabelled antibody enzyme (PAP) method, we observed few scattered S-100 and GFA positive cells in the pineal. The number, location and morphology of these cells suggest they are the pineal interstitial cells. This indicates that the interstitial cells are of neuroectodermal origin, possibly macroglial cells themselves.

The immunocytochemical localization for hydroxyindole O-methyltransferase (HIOMT), neuron-specific enolase (NSE) and S-100 protein in the bovine pineal gland is described in this paper. Most of the pinealocytes showed immunoreactivities to a HIOMT antiserum and an NSE antiserum, simultaneously. Neither HIOMT-nor NSE-immunoreactivity was observed in a few pinealocytes. On the other hand, dispersed interstitial cells (glial cells) were stained only by an S-100 protein antiserum. No evident HIOMT-immunoreactivity was observed in the retina and intestinal mucosa, where HIOMT has been suggested to occur.

Indoleamine levels in punches of the medial hypothalamus containing the suprachiasmatic nuclei (SCN) of 4-week-old cockerels were determined by HPLC-EC. Melatonin levels in punches were determined by radioimmunoassay (RIA). Daily rhythms of serotonin (5-HT) and of its metabolite 5-hydroxy-3-indoleacetic acid (5-HIAA) were observed; levels were higher at midnight than at mid-day. A daily rhythm with the same phase in punch melatonin content was also observed. Pinealectomy at 1 week after hatching abolished the 5-HIAA and melatonin rhythm in 4-week-old birds but did not abolish the 5-HT rhythm. Injections of melatonin (0.5 mg/kg) increased 5-HT, 5-HIAA and melatonin levels in the hypothalamic punches. These results indicate that circulating melatonin of pineal origin may act to increase 5-HT turnover and/or release in the SCN. They suggest a link between the circadian secretion of pineal melatonin and the regulation of 5-HT projections to the hypothalamus from the raphe nuclei in the brainstem of the chicken. We have previously shown that the rhythmic secretion of melatonin by the pineal is influenced by oscillators in the brain via the superior cervical ganglia8. The results reported here indicate that melatonin in turn may regulate brain oscillators, suggesting a neuroendocrine loop within the avian circadian system.

Investigations on the regulation of pineal melatonin synthesis in the Syrian hamster revealed distinct differences compared to this well-understood mechanism in rat. E.g., a circadian profile of pineal norepinephrine (NE) is absent, there is no β-adrenoceptor sensitivity during daytime and adrenergic receptor supersensitivity is not easily achieved. To elucidate the action of NE on pineal receptor sites, the effects of iontophoretic application of adrenergic compounds on spontaneous electrical discharge rates of pinealocytes were investigated during day- and nighttime. Following application of either NE, isoproterenol or clonidine, cells were activated, inhibited or not affected. Whereas about one-third of the units responded to iontophoretic application of sympathomimetics at daytime, the number of affected cells was doubled during the night. These results demonstrate the involvement of adrenoceptors in the regulation of circadian rhythms in electrophysiological properties of Syrian hamster pinealocytes. Since relatively few cells responded during daytime and inhibitory adrenergic mechanisms dominated at night, the classification of receptors by means of iontophoresis may provide a basis to explain the difficulty to influence pineal melatonin synthesis by sympathomimetics in the Syrian hamster.

The purpose of this study was to determine if serotonin (5-HT) is secreted by the pineal gland and also to define the parameters of its secretion. After radiolabelling the endogenous 5-HT pool, individual pineal glands were placed within perifusion chambers and stimulated with norepinephrine (NE). [3H]5-HT was rapidly released within 1 min of stimulation and secretion continued throughout the period of exposure to NE. The findings suggest that 5-HT, in addition to melatonin, is a hormone of the pineal gland.

Membrane properties and responsiveness to norepinephrine were studied in pinealocytes in tissue culture using both conventional and whole-cell patch-clamp techniques. Action potentials could be recorded in normal and tetrodotoxin-containing saline. Both outward and inward rectification were prominent. Under voltage-clamp, depolarizing voltage steps evoked time-dependent outward currents showing no inactivation over time. Hyperpolarizing steps evoked slowly developing inward currents which did not inactivate. If the holding potential was sufficiently negative, depolarizing steps also elicited a transient outward current. Iontophoresis of norepinephrine produced a hyperpolarization in 30% of cells tested.