Developmental maturation of pineal gland function: synchronized CREM inducibility and adrenergic stimulation.

The cAMP response element modulator (CREM) gene encodes multiple activators and repressors of cAMP-responsive transcription. Differential splicing generates a developmental switch in CREM function during spermatogenesis, while the use of an alternative promoter is responsible for the production of a cAMP-inducible transcriptional repressor, ICER (inducible cAMP early repressor). The ICER promoter is strongly inducible by cAMP because of the presence of four tandemly repeated cAMP response elements. Furthermore, ICER negatively autoregulates the ICER promoter activity, thus generating a feedback loop. CREM constitutes an early response gene of the cAMP pathway in several neuroendocrine cells. We have previously shown that CREM is highly expressed in the adult rat pineal gland at nighttime. Here, we show that the only additional site of rhythmic ICER expression within the photoneuroendocrine system is the lamina intercalaris. Ontogenetically, the ICER day-night switch and cAMP inducibility mature in the pineal gland at the end of the first postnatal week. Importantly, this correlates with the onset of melatonin synthesis and the establishment of functional adrenergic innervation. At this developmental phase we document a significant increase in protein kinase A levels, thus suggesting that ICER inducibility reflects a complete maturation of the cAMP-dependent signaling pathway at the nuclear level.

Molecular cloning and characterization of a rat A1-adenosine receptor that is widely expressed in brain and spinal cord.

An A1-adenosine receptor has been cloned from a rat brain cDNA library using a probe generated by the polymerase chain reaction. The cDNA encodes a protein of 327 amino acids which is 91% identical to a recently cloned dog A1-adenosine receptor (RDC7). Expression of the rat cDNA in COS-6M and NIH 3T3 cells resulted in ligand binding and functional activity characteristics of an A1-adenosine receptor that is coupled to inhibition of adenylyl cyclase. Examination of the distribution of A1-adenosine receptor mRNA by Northern blot analysis showed that it is highly expressed in brain, spinal cord, testis, and white adipose tissue. In situ hybridization studies revealed an extensive hybridization pattern in the central nervous system, with high levels in cerebral cortex, hippocampus, cerebellum, thalamus, brainstem, and spinal cord. The cloned A1-adenosine receptor may thus mediate many of the modulatory actions of adenosine in neural and endocrine systems.

Molecular cloning and expression of the cDNA for a novel A2-adenosine receptor subtype.

A novel adenosine receptor subtype has been cloned from a rat brain cDNA library using a probe generated by the polymerase chain reaction. The cDNA, designated RFL9, encodes a protein of 332 amino acids. The structure of RFL9 is most similar to that of the recently cloned rat A2-adenosine receptor, with a sequence identity of 73% within the presumed seven transmembrane domains. Expression of RFL9 in COS-6M cells resulted in ligand binding and functional activity characteristics of an adenosine receptor that is coupled positively to adenylyl cyclase. Examination of the tissue distribution of RFL9 mRNA by Northern blot analysis showed a restricted distribution with highest levels expressed in large intestine, cecum, and urinary bladder; this pattern was distinct from that of either the A1- or A2-adenosine receptor mRNAs. In situ hybridization studies of RFL9 mRNA showed no specific hybridization pattern in brain, but a hybridization signal was readily observed in the hypophyseal pars tuberalis. Thus, RFL9 encodes a novel A2-adenosine receptor subtype.

Of rodents and ungulates and melatonin: creating a uniform code for darkness by different signaling mechanisms.

Melatonin synthesis in the mammalian pineal gland is one of the best investigated output pathways of the circadian clock because it can be readily measured and is tightly regulated by a clearly defined input, the neurotransmitter norepinephrine. In this system, a regulatory scenario was deciphered that is centered around the cyclic AMP pathway but shows peculiar species-specific differences. In rodents, the cyclic AMP-mediated, temporally sequential up-regulation of two transcription factors, the activator CREB (cyclic AMP-responsive element-binding protein) and the inhibitor ICER (inducible cyclic AMP-dependent early repressor), is the core mechanism to determine rhythmic accumulation of the mRNA encoding for the rate-limiting enzyme in melatonin synthesis, the arylalkylamine N-acetyltransferase (AA-NAT). Thus, in rodents, the regulation of melatonin synthesis bears an essential transcriptional component, which, however, is flanked by posttranscriptional mechanisms. In contrast, in ungulates, and possibly also in primates, AA-NAT appears to be regulated exclusively on the posttranscriptional level. Here, increasing cyclic AMP levels inhibit the breakdown of constitutively synthesized AA-NAT protein by proteasomal proteolysis, leading to an elevated enzyme activity. Thus, self-restriction of cellular responses, as a reaction to external cues, is accomplished by different mechanisms in pinealocytes of different mammalian species. In such a temporally gated cellular adaptation, transcriptionally active products of clock genes may play a supplementary role. Their recent detection in the endogenously oscillating nonmammalian pineal organ and, notably, also in the slave oscillator of the mammalian pineal gland underlines that the mammalian pineal gland will continue to serve as an excellent model system to understand mechanisms of biological timing.

Melatonin receptors in human hypothalamus and pituitary: implications for circadian and reproductive responses to melatonin.

Two major physiological roles for the pineal hormone melatonin (MEL) have been identified in vertebrates: the hormone influences circadian rhythmicity and regulates seasonal responses to changes in day length. These effects of MEL are thought to be due to interaction with specific, high affinity MEL receptors in the suprachiasmatic nucleus (SCN) and hypophysial pars tuberalis (PT), respectively. Using the ligand 2-[125I]iodo-MEL ([125I]MEL), we examined putative MEL receptors in these regions in human and monkey tissue specimens by in vitro autoradiography. Specific, high affinity [125I]MEL-binding sites (Kd, 53.3 +/- 13.0 pM) were consistently observed in the human SCN. In contrast, specific [125I]MEL binding was detectable in the PT of only one of the eight human specimens examined. Specific [125I]MEL binding was also detected in the pars distalis of several subjects, but with an inconsistent distribution. In rhesus monkey tissue, MEL receptors were readily detected in the SCN and, as in all other seasonally breeding species examined to date, in the PT. The relative absence of MEL receptors from the human PT suggests that neuroendocrine responses to MEL in humans may occur by fundamentally different mechanisms than those that underlie the photoperiodic regulation of reproduction in seasonally breeding species.

Exploration of a novel environment leads to the expression of inducible transcription factors in barrel-related columns.

Tactile information acquired through the vibrissae is of high behavioral relevance for rodents. Numerous physiological studies have shown adaptive plasticity of cortical receptive field properties due to stimulation and/or manipulation of the whiskers. However, the cellular mechanisms leading to these plastic processes remain largely unknown. Although genomic responses are anticipated to take place in this sequel, virtually no data so far exist for freely behaving animals concerning this issue. Thus, adult rats were placed overnight in an enriched environment and most of them were also subjected to clipping of different sets of whiskers. This type of stimulation led to a specific and statistically significant increase in the expression of the protein products of the inducible transcription factors c-Fos, JunB, inducible cyclic-AMP early repressor and Krox-24 (also frequently named Zif268 or Egr-1), but not c-Jun. The response was found in columns of the barrel cortex corresponding to the stimulated vibrissae; it displayed a layer-specific pattern. However, no induction of transcription factors was observed in the subcortical relay stations of the whisker-to-barrel pathway, i.e. the trigeminal nuclei and the ventrobasal complex. These results strongly suggest that a coordinated transcriptional response is initiated in the barrel cortex as a consequence of processing of novel environmental stimuli.

The pineal organ, its hormone melatonin, and the photoneuroendocrine system.

The vertebrate pineal organ rhythmically synthesizes and secretes melatonin during nighttime and forms an essential component of the photoneuroendocrine system which allows humans and animals to measure and keep the time. Regulation of the melatonin biosynthesis depends on signals from photoreceptors perceiving and transmitting environmental light stimuli and endogenous oscillators generating a circadian rhythm which is independent from any environmental time cue (zeitgeber). In nonmammalian species the photoreceptors responsible for regulating melatonin biosynthesis reside within the pineal organ itself. In several nonmammalian species (e.g., lamprey, zebra fish, house sparrow, chicken) the pineal organ is also capable of generating circadian rhythms and thus serves all key functions of the photoneuroendocrine system: photoreception, endogenous rhythm generation, and production of neurohormones. These may even be accomplished by a single "photoneuroendocrine" cell. In mammals the pineal organ has lost both the direct light sensitivity and the capacity of generating circadian rhythms, and melatonin biosynthesis is regulated by retinal photoreceptors and a circadian oscillator located in the suprachiasmatic nucleus of the hypothalamus. Due to this spatial separation the photoneuroendocrine system of mammals comprises neuronal and neuroendocrine pathways which interconnect its components. The neuronal pathways involve circuits of both the central and the peripheral nervous systems, and as an important final link noradrenergic sympathetic nerve fibers. The suprachiasmatic nucleus appears as a major target of melatonin in mammals. The pineal hormone may thus be involved in a feedback loop of the mammalian photoneuroendocrine system. The present comparative contribution considers, after a short survey of classical findings on the phylogenetic development and the gross anatomy of the pineal complex, cytoevolutionary and cell biological aspects of the various types of pinealocytes as well as the afferent and efferent innervation of the pineal organ (pinealofugal and pinealopetal neuronal pathways). Moreover, emphasis is placed on receptor mechanisms, second messenger systems (Ca2+ and cyclic AMP), transcription factors (e.g, CREB and ICER), and their roles for regulation of melatonin biosynthesis. Finally, the action, targets, and receptors of melatonin are dealt with. The synoptic approach of this contribution, which combines anatomical and ultrastructural findings with cell and molecular biological results, confirms the functional significance of the melatonin-synthesizing pineal organ as an important component of the photoneuroendocrine system and stresses the importance of this organ as a model to study signal transduction mechanisms both in photoreceptors and in neuroendocrine cells.

Melatonin: a clock-output, a clock-input.

In mammals, the circadian system is comprised of three major components: the lateral eyes, the hypothalamic suprachiasmatic nucleus (SCN) and the pineal gland. The SCN harbours the endogenous oscillator that is entrained every day to the ambient lighting conditions via retinal input. Among the many circadian rhythms in the body that are driven by SCN output, the synthesis of melatonin in the pineal gland functions as a hormonal message encoding for the duration of darkness. Dissemination of this circadian information relies on the activation of melatonin receptors, which are most prominently expressed in the SCN, and the hypophyseal pars tuberalis (PT), but also in many other tissues. A deficiency in melatonin, or a lack in melatonin receptors should therefore have effects on circadian biology. However, our investigations of mice that are melatonin-proficient with mice that do not make melatonin, or alternatively cannot interpret the melatonin message, revealed that melatonin has only minor effects on signal transduction processes within the SCN and sets, at most, the gain for clock error signals mediated via the retino-hypothalamic tract. Melatonin deficiency has no effect on the rhythm generation, or on the maintenance of the oscillation. By contrast, melatonin is essential for rhythmic signalling in the PT. Here, melatonin acts in concert with adenosine to elicit rhythms in clock gene expression. By sensitizing adenylyl cyclase, melatonin opens a temporally-restricted gate and thus lowers the threshold for adenosine to induce cAMP-sensitive genes. This interaction, which determines a temporally precise regulation of gene expression, and by endocrine-endocrine interactions possibly also pituitary output, may reflect a general mechanism by which the master clock in the brain synchronizes clock cells in peripheral tissues that require unique phasing of output signals.

Clock gene protein mPER1 is rhythmically synthesized and under cAMP control in the mouse pineal organ.

The mammalian clock gene Per1 is an important element of endogenous oscillators that control daily rhythms in central and peripheral tissues. Although such autonomous clock function is lost in the mammalian pineal gland during evolution, mPer1 mRNA and mPER1 protein were found to be strongly elevated in the mouse pineal organ during the dark period compared to daytime values. In vitro studies showed that mPer1 mRNA and mPER1 protein in mouse pineal gland are induced following the activation of a signalling pathway of fundamental importance for pineal physiology, the norepinephrine/cAMP/phosphoCREB cascade. mPER1 may function in the mouse pineal gland as a time-measuring molecule to participate in regulating rhythmic cellular responses in vivo.

Melatonin limits transcriptional impact of phosphoCREB in the mouse SCN via the Mel1a receptor.

In the mouse, activity phase-shifts of the endogenous clock in the suprachiasmatic nucleus (SCN) are associated with phosphorylation of the transcription factor Ca2+/cAMP responsive element binding protein (CREB). CREB phosphorylation is induced by the retino-hypothalamic transmitter pituitary adenylate cyclase-activating polypeptide (PACAP). As detected by immunohistochemistry in SCN slices from wild-type mice, melatonin completely blocked PACAP-stimulated CREB phosphorylation at low concentrations (1 nM). In Mel1a melatonin receptor-deficient mice, the PACAP-induced CREB phosphorylation was inhibited only at melatonin concentrations of 100 nM. This inhibition was, however, blunted by blocking the Mel1b melatonin receptor. Thus, melatonin modulates PACAP-mediated retinal stimuli for clock entrainment primarily via the Mel1a melatonin receptor through molecular interaction within the cAMP-signalling pathway.

Ontogeny of a diurnal rhythm in arylalkylamine-N-acetyltransferase mRNA in rat pineal gland.

Melatonin synthesis in the pineal gland of adult rats is linked to cAMP-dependent transcriptional and post-transcriptional regulatory mechanisms affecting its rate-limiting enzyme, the arylalkylamine-N-acetyltransferase (AA-NAT). During development of the pineal gland, neuronal control gains access to the earlier matured cAMP-signaling pathway to shape the day-night rhythm in AA-NAT enzymatic activity. By semiquantitative in situ hybridization we analyzed if the developmental onset of a rhythmic AA-NAT activity is correlated to a temporally parallel onset in AA-NAT transcription. We found that AA-NAT mRNA levels in rat pineal gland become rhythmic at postnatal day 5. Thus, AA-NAT gene transcription in rat pineal gland starts to show day-night differences shortly prior to the appearance of a rhythmic AA-NAT activity.

Light-induced expression of transcription factor ICER (inducible cAMP early repressor) in rat suprachiasmatic nucleus is phase-restricted.

The mammalian hypothalamic suprachiasmatic nucleus (SCN) harbors an intrinsic circadian oscillator driving a variety of endogenous rhythms. SCN activity is entrained to environmental lighting conditions by photic information from the retina. Light-induced phase shifts involve cAMP and Ca2+ as second messengers and are linked to transcriptional and translational processes. Using in situ hybridization with a cDNA fragment of the cAMP responsive element modulator (CREM) isoform inducible cAMP early repressor (ICER) we demonstrate a light-induced upregulation of CREM mRNA in rat SCN during the second half of the night. Notably, ICER is the only member of the CREM family which is transcriptionally inducible. We suggest that a stimulus-induced upregulation of ICER expression can inhibit cAMP-inducible genes in rat SCN via its trans-repressing potency.

Signal transduction in the rodent pineal organ. From the membrane to the nucleus.

The rodent pineal organ transduces a photoneural input into a hormonal output. This photoneuroendocrine transduction leads to highly elevated levels of the hormone melatonin at night-time which serves as a message for darkness. The melatonin rhythm depends on transcriptional, translational and posttranslational regulation of the arylalkylamine-N-acetyltransferase, the key enzyme of melatonin biosynthesis. These regulatory mechanisms are fundamentally linked to two second messenger systems, namely the cAMP- and the Ca(2+)-signal transduction pathways. Our data gained by molecular biology, immunohistochemistry and single-cell imaging demonstrate a time- and substance-specific activation of these signaling pathways and provide a framework for the understanding of the complex signal transduction cascades in the rodent pineal gland which in concert not only regulate the basic profile but also fine-tune the circadian rhythm in melatonin synthesis.

Pineal gene expression: dawn in a dark matter.

The mammalian pineal gland serves as a neuroendocrine interface to convert environmental lighting conditions into a humoral message, the nocturnally elevated synthesis of melatonin. Regulation and fine tuning of the circadian melatonin production in response to external cues requires complex interactions of transsynaptic signalling. These requirements are fulfilled by a high degree of plasticity on all levels between receptor activation and cellular response. Many receptors on pinealocytic membranes and enzymes involved in melatonin synthesis are linked to the second messenger cAMP. Crosstalk between second and third messengers converges in the pineal gland--as in other tissues--eventually on a modulated activity of transcription factors. Of fundamental importance for genes involved in the transsynaptic signalling to create a circadian profile in melatonin synthesis is the cAMP-inducible promoter element, the CRE (cAMP responsive element). Indeed, the CRE is shared by many pineal genes that are of physiological importance. Recently, the deciphering of molecular determinants regulating expression of cAMP-inducible genes in the mammalian pineal gland, like NAT, c-jun, or the beta-adrenergic receptor suggests a modulation in their transcription by a dual regulatory mechanism: posttranslational activation of the early third messenger CREB (cAMP responsive element binding protein) stimulates, cis-acting cAMP-induced transcriptional upregulation of the late third messenger ICER (inducible cAMP early repressor) inhibits genes with a CRE.

Analysis of cell signalling in the rodent pineal gland deciphers regulators of dynamic transcription in neural/endocrine cells.

In neurons, a temporally restricted expression of cAMP-inducible genes is part of many developmental and adaptive processes. To understand such dynamics, the neuroendocrine rodent pineal gland provides an excellent model system as it has a clearly defined input, the neurotransmitter norepinephrine, and a measurable output, the hormone melatonin. In this system, a regulatory scenario has been deciphered, wherein cAMP-inducible genes are rapidly activated via the transcription factor phosphoCREB to induce transcriptional events necessary for an increase in hormone synthesis. However, among the activated genes is also the inhibitory transcription factor ICER. The increasing amount in ICER protein leads ultimately to the termination of mRNA accumulation of cAMP-inducible genes, including the gene for the Aa-nat that controls melatonin production. This shift in ratio of phosphoCREB and ICER levels that depends on the duration of stimulation can be interpreted as a self-restriction of cellular responses in neurons and has also been demonstrated to interfere with cellular plasticity in many non-neuronal systems.

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.

Antisense experiments reveal molecular details on mechanisms of ICER suppressing cAMP-inducible genes in rat pinealocytes.

In the rat pineal gland neuronal signals determine the rhythmic synthesis of the hormone melatonin. Norepinephrine (NE) is the principal neurotransmitter that drives hormone synthesis by activating the cAMP signaling pathway. This activation depends on transcriptional and posttranscriptional regulatory mechanisms. The cAMP-dependent transcriptional regulation of the rate-limiting enzyme of melatonin synthesis, arylalkylamine-N-acetyltransferase (AA-NAT) involves the activating transcription factor (TF) CREB and the inhibitory TF ICER. By silencing elements of this cAMP-dependent neuroendocrine transduction cascade we wished to gain further insight into the role of ICER in the regulation of gene expression in rat pineal gland. Inhibition of specific kinases in primary pinealocyte cultures showed that ICER induction depends pivotally on the activation of cAMP-dependent protein kinase II. Eliminating ICER's impact by transfecting antisense constructs into pinealocytes revealed a predominant beta-adrenergic mechanism in regulating a cotransfected CRE-inducible reporter gene and notably, also the endogenous AA-NAT gene. Deciphering molecular details of the cAMP-dependent gene expression in mammalian pinealocytes provides a basis for understanding the general architecture of this signaling pathway that serves adaptive processes ubiquitously in the organism.

Norepinephrine-induced phosphorylation of the transcription factor CREB in isolated rat pinealocytes: an immunocytochemical study.

In the present study we investigated whether norepinephrine, which stimulates melatonin biosynthesis in the mammalian pineal organ, causes phosphorylation of the cyclic AMP responsive element binding protein (CREB) in rat pinealocytes. Cells isolated from the pineal organ of adult male rats and cultured on coated coverslips were treated with norepinephrine, beta- or alpha 1-adrenergic agonists for 12, 5, 10, 20, 30, 60 or 300 min and then immunocytochemically analyzed with an antibody against phosphorylated CREB (p-CREB). Treatment with norepinephrine or beta-adrenergic agonists resulted in a similar, time-dependent induction of p-CREB immunoreactivity, exclusively found in cell nuclei. The alpha 1-adrenergic agonist phenylephrine did not induce p-CREB immunoreactivity at low doses (0.1 microM) or when high doses (10 microM) were applied in combination with a beta-antagonist (propranolol, 0.1 microM). This indicates that induction of CREB phosphorylation is elicited by beta-adrenergic receptor stimulation. The response was first seen after 10 min and reached a maximum after 30 to 60 min when more than 90% of the cells displayed p-CREB immunoreactivity. The intensity of the p-CREB immunoreactivity showed marked cell-to-cell variation, but nearly all immunoreactive cells were identified as pinealocytes by double-labeling with an antibody against the S-antigen, a pinealocyte-specific marker. The results show that norepinephrine stimulation induces p-CREB immunoreactivity by acting upon beta-adrenergic receptors in virtually all rat pinealocytes. The findings support the notion that phosphorylation of CREB is a rather rapid and uniform response of pinealocytes to noradrenergic stimulation and thus is an important link between adrenoreceptor activation and subsequent gene expression in the rat pineal organ.

Control of CREB phosphorylation and its role for induction of melatonin synthesis in rat pinealocytes.

Phosphorylation of the transcription factor CREB appears as an important step in the signal transduction cascade that activates melatonin biosynthesis in the mammalian pineal organ. We have studied the mechanisms causing CREB phosphorylation by immunocytochemical and immunochemical demonstration of phosphorylated CREB (pCREB) in isolated, immunocytochemically identified rat pinealocytes kept in vitro and in the rat pineal organ in situ. Norepinephrine (NE), the most potent stimulator of the melatonin biosynthesis was shown to induce pCREB immunoreaction (i.r.) in the vast majority of pinealocytes in a time- and dose-dependent manner. This response was elicited by stimulation of beta-adrenergic receptors resulting in an increase in the intracellular cAMP concentration. Activation of alpha 1-adrenergic receptors that causes a rise in intracellular calcium via stimulation of intracellular stores and subsequent increase in calcium influx did not evoke pCREB ir on its own and did not potentiate the beta-adrenergic response. VIP and PACAP that activate the melatonin biosynthesis to a lesser extent than NE induced pCREB ir in only 50-60% of the pinealocytes. Immunoblotting showed that a protein of 43 kDa corresponding to CREB accounts for the pCREB ir and confirmed that VIP and PACAP are less effective in inducing CREB phosphorylation than NE. The amount of total (phosphorylated and unphosphorylated) CREB was not changed upon stimulation of the cells with NE, VIP or PACAP. In an attempt to identify the protein kinase catalyzing CREB phosphorylation in rat pinealocytes, the cAMP-dependent protein kinases (cAK) present in the rat pineal were identified with the use of antibodies recognizing different catalytic and regulatory subunits. Application of cAK agonists and antagonists showed that the cAK type II is responsible for CREB phosphorylation. Correlations between the melatonin concentration in the medium and the CREB phosphorylation in pinealocytes revealed a tight connection between these two parameters. Phosphorylation of CREB appears important for the stimulation of melatonin biosynthesis also under natural conditions because our investigations of whole pineal organs taken from rats during different time points of the 24 h light-dark cycle revealed a strong induction of pCREB ir in the first part of the night.

Rhythmic variation in beta1-adrenergic receptor mRNA levels in the rat pineal gland: circadian and developmental regulation.

In the rat pineal gland noradrenaline is released in large quantities from sympathetic nerve endings at the onset of darkness, thereby driving rhythmic melatonin synthesis with elevated levels at night-time. Upon release, noradrenaline interacts with postsynaptic beta1-adrenergic receptors to activate the cyclic AMP signalling pathway. Well characterized third messengers of this signalling cascade affect cyclic AMP-inducible genes that are crucially involved in initiation, maintenance and termination of hormone production. Among these third messengers are CREB (cyclic AMP responsive element binding protein) as an activating and ICER (inducible cyclic AMP early repressor) as an inhibitory transcription factor. Because a cyclic AMP-inducible promoter element is present on the beta1-adrenergic receptor gene, the expression of the receptor itself may be under control of the cyclic AMP-signalling pathway. By in situ hybridization, Northern blot analysis and RT-PCR we demonstrate a day/night rhythm in beta1-adrenergic receptor mRNA in the rat pineal gland with elevated levels during the dark period. As this rhythm persists, under constant darkness but is abolished upon removal of the sympathetic innervation, it is truly circadian. A marked day/night difference in the levels of beta1-adrenergic receptor mRNA becomes evident only after postnatal day 10, coinciding with the appearance of a functional cyclic AMP signalling pathway in the rat pineal gland. Furthermore, targeting ICER expression by transfection of pinealocytes with an antisense ICER construct, clearly indicates that the levels of the beta1-adrenergic receptor mRNA are regulated by the cyclic AMP-signalling pathway in a feedback mechanism.