Melatonin induces gene-specific effects on rhythmic mRNA expression in the pars tuberalis of the Siberian hamster (Phodopus sungorus)

In mammals, circadian and photoperiodic information is encoded in the pineal melatonin signal. The pars tuberalis (PT) of the pituitary is a melatonin target tissue, which transduces photoperiodic changes and drives seasonal changes in prolactin secretion from distal lactotroph cells. Measurement of photoperiodic time in the PT is believed to occur through melatonin dependent changes in clock gene expression, although it is unclear whether the PT should be considered a melatonin sensitive peripheral oscillator. We tested this hypothesis in the Siberian hamster (Phodopus sungorus) firstly by investigating the effects of melatonin injection, and secondly by determining whether temporal variation in gene expression within the PT persists in the absence of a rhythmic melatonin signal. Hamsters preconditioned to long days were treated with melatonin during the late light phase, to advance the timing of the nocturnal melatonin peak, or placed in constant light for one 24 h cycle, thereby suppressing endogenous melatonin secretion. Gene expression in the PT was measured by in situ hybridization. We show that melatonin rapidly induces cry1 mRNA expression without the need for a prolonged melatonin-free interval, acutely inhibits mt1 expression, advances the timing of peak rev-erb alpha expression and modulates per1 expression. With the exception of cry1, these genes continue to show temporal changes in expression over a first cycle in the absence of a melatonin signal. Our data are consistent with the hypothesis that the hamster PT contains a damped endogenous circadian oscillator, which requires a rhythmic melatonin signal for long-term synchronization.     

The human MT1 melatonin receptor stimulates cAMP production in the human neuroblastoma cell line SH-SY5Y cells via a calcium-calmodulin signal transduction pathway

Melatonin regulates circadian and seasonal physiology via melatonin receptors expressed in the brain. However, little is known about the signal transduction mechanisms that mediate the action of melatonin in neuronal cells. To begin to address this issue, we expressed the human MT(1) receptor in the human neuroblastoma SH-SY5Y cell line. In this cell line, melatonin acutely stimulated cAMP synthesis through a calcium-calmodulin dependent pathway. This stimulatory effect was independent of an interaction with G(i) or G(s) G proteins and dependent upon internal calcium stores. Melatonin also potentiated forskolin-activated cAMP synthesis. Differentiation of the neuroblastoma cells with retinoic acid to the neuronal phenotype did not alter the ability of melatonin to acutely stimulate cAMP. These data may be relevant to the neuronal action of melatonin and highlight the importance of the cellular context of expression of melatonin and other G protein-coupled receptors.     

Melatonin modulates spike coding in the rat suprachiasmatic nucleus

The effects of the application of melatonin in vitro on the electrophysiological activity of suprachiasmatic neurones were characterised using novel measures of coding based on the analysis of interspike intervals. Perfusion of 1 nM melatonin in vitro (n = 53) had no consistent effect on mean spike frequency (Wilcoxon's sign rank, z = -0.01, P = 0.989), but increased the irregularity of firing (Student's paired t-test, t = -3.02, P = 0.004), as measured by the log interval entropy, and spike patterning (z = -3.43, P < 0.001), as measured by the mutual information between adjacent log intervals. Intracellular recordings in vitro in current clamp mode showed that 1 nM melatonin significantly hyperpolarised (n = 11, z = -2.35, P = 0.019) those cells that showed 'rebound' spikes upon termination of a hyperpolarising current pulse. Grouping all cells together (n = 27), melatonin application decreased the duration of the afterhyperpolarisation (z = -2.49, P = 0.013) and increased the amplitude of the depolarising afterpotential (z = -2.71, P = 0.007). The effects of melatonin seen in vitro from extracellular recordings on interspike interval coding were consistent with the changes in spike shape seen from intracellular recordings. A melatonin-induced increase in the size of the depolarising afterpotential of suprachiasmatic cells might underlie the increased irregularity of spike firing seen during the subjective night time. The method of analysis demonstrated a difference in spike firing that is not revealed by frequency alone and is consistent with the presence of a melatonin-induced depolarising current.     

Localization of circadian clock protein BMAL1 in the photoperiodic signal transduction machinery in Japanese quail

The circadian clock is a fundamental property of living organisms and is involved in seasonal (photoperiodic) time measurement. Among vertebrates, birds have multiple circadian pacemakers in the eye, the pineal gland, and the suprachiasmatic nucleus (SCN), and have highly sophisticated photoperiodic mechanisms. However, because the removal of these circadian pacemakers fails to abolish the photoperiodic response, the existence of another “photoperiodic clock” has been suggested. Recent studies have revealed that the mediobasal hypothalamus (MBH) and the adjacent pars tuberalis (PT) of the pituitary gland constitute key components of the photoperiodic signal transduction machinery. In the present study, we generated a polyclonal antibody against the chicken circadian clock protein BMAL1 to examine BMAL1 distribution in the Japanese quail brain by using immunohistochemistry. BMAL1‐like immunoreactivity (lir) was confirmed in the pineal gland and the medial SCN, which are critical circadian pacemakers. We also observed strong immunoreactivity in the MBH, including the ependymal cells (ECs), the infundibular nucleus (IN), the median eminence (ME), and the adjacent PT. Furthermore, semiquantitative analysis suggested that BMAL1‐lir shows daily fluctuation in these regions. It is possible that circadian clocks in the photoperiodic signal transduction machinery such as the PT and the EC may be involved in the regulation of photoperiodism. J. Comp. Neurol. 517:397–404, 2009. © 2009 Wiley‐Liss, Inc.     

Brain and Pituitary Melatonin Receptors in Male Rat during Post-Natal and Pubertal Development and the Effect of Pinealectomy and Testosterone Manipulation

Using quantitative autoradiography, melatonin receptors have been studied during post-natal and pubertal development of the rat in 2 brain and 2 pituitary structures. In the pars distalis of anterior pituitary, melatonin receptors decrease gradually in density after birth and disappear in 30 day-old animals. In contrast melatonin binding is only expressed in the paraventricular nuclei of the thalamus at the age of 21–23 days and is always present in adult animals. In the suprachiasmatic nuclei and in the pars tuberalis of the pituitary, melatonin receptor density decreases after birth, remains stable for approximately 1 month and increases again at puberty to reach the birth values in the adult. This increase was absent in pinealectomized and in castrated animals but present in castrated animals receiving testosterone suggesting that it depends upon circulating testosterone and melatonin levels. These results show that melatonin receptors are differentially regulated during post-natal development in each of the 4 structures studied, and that melatonin and testosterone are 2 factors which could be involved in the regulation of melatonin receptor density in the suprachiasmatic nuclei and pars tuberalis.     

Differential Seasonal Regulation of Melatonin Receptor Density in the Pars Tuberalis and the Suprachiasmatic Nuclei: A Study in the Hedgehog (Erinaceus europaeus, L.)

Using quantitative autoradiography, we have studied the seasonal changes of high affinity melatonin receptor density in both the SCN and PT of the hedgehog, a seasonal breeder and an hibernator. Animals in 3 different physiological states were studied: sexually active animals, and sexually inactive animals during the hibernation period, being then either euthermic or hypothermic. In sexually active animals, B_maax were 75.8 ± 7.1 fmol/mg protein in PT and 9.1 ± 0.5 fmol/mg protein in SCN; and Kd values were: 94 ± 22 pM in the PT and 101 ± 15 pM in the SCN. This specific binding was strongly decreased in the PT of sexually inactive animals. Moreover, this decrease was significantly stronger in hypothermic than in euthermic hedgehogs. Saturation studies and Scatchard analysis revealed that the observed decrease in the PT resulted from change in the B_max but not in the Kd, B_max values being respectively 56.4 ± 5.9 and 29.5 ± 1.9 fmol/mg protein in euthermic and hypothermic sexually at rest animals. In none of the different physiological states, did the density of melatonin receptors of the SCN show any changes, B_maax values being respectively 9.8 ± 0.5 and 9.8 ± 0.4 fmol/mg protein in euthermic and hypothermic sexually at rest animals. This shows for the first time a tissue-specific regulation of melatonin receptor density occurring in the PT but not in the SCN. Furthermore, this decrease of binding in the PT is correlated with both sexual inactivity and hibernation period. This strongly suggests that the mediation of the photoperiodic effect on seasonal functions like seasonal hypothermia and reproduction involves an effect of melatonin on the PT rather than on the SCN.     

Melatonin Receptors on Ovine Pars Tuberalis: Characterization and Autoradiographicai Localization

The functional significance of the pars tuberalis (PT) of the mammalian adenohypophysis has remained an enigma (1, 2). One view of its function is that it acts as an auxiliary gland to support the endocrine role of the pars distalis (PD) (2), as it has been shown to contain immunocytochemically identifiable thyrotrophs and gonadotrophs (1). Many of the cells of the PT are, however, ultrastructurally unique suggesting an independent function for this tissue. Our recent demonstration that the PT of the rat is a major binding site for the ligand iodomelatonin lends further support to this idea (3). We have utilized the highly specific ligand [¹²⁵l]melatonin, and have demonstrated that it binds exclusively, with very high affinity, to the PT but not the PD of the adult sheep adenohypophysis. These findings support the conclusion that the PT has a distinct role in relation to melatonin action and seasonal reproduction.     

Evidence for Multiple Forms of Melatonin Receptor-G-Protein Complexes by Solubilization and Gel Electrophoresis

The daily production of melatonin from the pineal gland influences circadian and seasonal behaviour and physiology. To further understand how melatonin may function, it is important to characterize the receptor and signal transduction systems. Using the detergent digitonin, we were able to solubilize the receptor from the ovine pars tuberalis (PT) membrane. The receptor was isolated as a complex associated with its heterotrimeric G-protein. In the solubilized state, pre-bound ¹²⁵l-2-iodomelatonin was stable at 4°C, but was displaceable by GTPγS. The receptor-G-protein complex could be separated by molecular mass using native polyacrylamide gel electrophoresis. We demonstrate that the receptor-complex has a molecular mass of 525 kDa and differs from solubilized receptor-complexes isolated from either the lizard brain, chicken brain or the ovine hippocampus. Furthermore the receptor complex isolated from the hippocampus had the lowest molecular mass of these tissues (365 kDa) and was found not to be sensitive to GTPγS. This may indicate the existence of a distinct non-G-protein coupled form of the receptor.     

Does a Melatonin‐Dependent Circadian Oscillator in the Pars Tuberalis Drive Prolactin Seasonal Rhythmicity?

The pars tuberalis (PT) of the adenohypophysis expresses a high density of melatonin receptors and is thought to be a crucial relay for the actions of melatonin on seasonal rhythmicity of prolactin secretion by the pars distalis (PD). In common with the suprachiasmatic nucleus of the hypothalamus and most other peripheral tissues, the PT rhythmically expresses a range of 'clock genes'. Interestingly, this expression is highly dependent upon melatonin/photoperiod, with several aspects unique to the PT. These observations led to the establishment of a conceptual framework for the encoding of seasonal timing in this tissue. This review summarises current knowledge of the morphological, functional and molecular aspects of the PT and considers its role in seasonal timing. The strengths and weaknesses of current hypotheses that link melatonin action in the PT to its seasonal effect on lactotrophs of the PD are discussed and alternative working hypotheses are suggested.     

Photic regulation of mt1 melatonin receptors in the Siberian hamster pars tuberalis and suprachiasmatic nuclei: involvement of the circadian clock and intergeniculate leaflet

In the Siberian hamster suprachiasmatic nuclei and pars tuberalis of the pituitary, high affinity mt1 melatonin receptors are present. We have previously shown that night applied light pulse induced an increase in mt1 mRNA expression in the suprachiasmatic nuclei of this species, independently of the endogenous melatonin. Here, we report the photic regulation of melatonin receptor density and mRNA expression in the suprachiasmatic nuclei and pars tuberalis of pinealectomized Siberian hamsters and the implication in this control of either the circadian clock or the intergeniculate leaflet. The results show that: (1) A 1-h light pulse, delivered during the night, induces a transitory increase in mt1 mRNA expression in the suprachiasmatic nuclei and pars tuberalis. After 3 h this increase has totally disappeared (suprachiasmatic nuclei) or is greatly reduced (pars tuberalis). (2) The melatonin receptor density, in the suprachiasmatic nuclei, is not affected by 1 or 3 h of light, while it is strongly increased in the pars tuberalis. (3) In hamsters kept in constant darkness, the mt1 mRNA rise is gated to the subjective night in the suprachiasmatic nuclei and pars tuberalis. In contrast, the light-induced increase in melatonin binding is also observed in the subjective day in the pars tuberalis. (4) intergeniculate leaflet lesion totally inhibits the mt1 mRNA expression rise in the suprachiasmatic nuclei, while it has no effect on the light-induced increase in mt1 mRNA in the pars tuberalis. However, the light-induced increase in melatonin receptor density is totally prevented by the intergeniculate leaflet lesion in the pars tuberalis. These results show that: (1) the photic regulations of mt1 mRNA expression and receptor density are independent of each other in both the suprachiasmatic nuclei and pars tuberalis; and (2) the circadian clock and the intergeniculate leaflet are implicated in the photic regulation of melatonin receptors but their level of action differs totally between the suprachiasmatic nuclei and pars tuberalis.     

Photoperiodically-lnduced Cycles in the Secretion of Prolactin in Hypothalamo-Pituitary Disconnected Rams: Evidence for Translation of the Melatonin Signal in the Pituitary Gland

Long term changes in the secretion of prolactin were monitored in groups of hypothalamo-pituitary disconnected rams (HPD rams, n = 8) and control rams (HPD sham-operated and unoperated, n = 8) while exposed to an artificial lighting regimen of alternating 16-weekly periods of long days (16L : 8D) and short days (8L : 16D) for 72 weeks, and during a treatment with subcutaneous constant-release implants of melatonin under long days. The HPD rams showed all the clinical characteristics of complete pituitary disconnection (diabetes insipidus, gonadal regression and slight obesity), and were unresponsive to a range of provocation tests (exposure to a barking sheep dog, cannulation of the jugular vein, injection of serotonin and NMDA) which caused acute changes in the blood plasma concentrations of prolactin in the controls. Nevertheless, there was a clearly defined cycle in the blood concentrations of prolactin in the HPD rams related to the imposed lighting regimen with values 10-fold higher under long days compared to short days (HPD mean ± SEM: 90.1 ± 24.7 vs 9.4 ± 2.0 μl, long vs short day respectively, P < 0.001). The temporal pattern was very similar to that observed in the controls, although the concentrations of prolactin were higher in the HPD rams and more variable (control mean ± SEM: 55.6 ± 3.6 vs 3.0±0.5 μl, long vs short day, P < 0.001). There was a corresponding cycle in the growth and moulting of the wool in the HPD rams consistent with a biological response to the photoperiodically-induced changes in the secretion of prolactin. The diurnal rhythm in the blood concentrations of prolactin was absent in the HPD rams, but there was a normal rhythm in the secretion of melatonin. The treatment of the animals with constant-release implants of melatonin under long days caused a marked decrease in the blood concentrations of prolactin in both the HPD and control rams. The overall conclusion is that the endogenously generated daily melatonin signal which encodes daylength acts directly in the pituitary gland to mediate the effects of photo-period on the secretion of prolactin. The photo-period transduction pathway thus by-passes the hypothalamus.     

Bilateral Lesions of the Suprachiasmatic Nuclei Alter the Nocturnal Melatonin Secretion in Sheep

The hypothalamic suprachiasmatic nuclei (SCN) constitute both the biological clock of many circadian rhythms, and the first relay in the transmission of light cues from the retina to the pineal gland, which releases, via nocturnal melatonin secretion, an endocrine expression of the daylength. The aim of the present work was to investigate the precise role of the SCN in the entrainment of the nocturnal rhythm of melatonin (MEL) in sheep. Bilateral lesions of the SCN were performed via a transsinusal surgical approach in 10 adult rams submitted to a constant photoperiod (16L:8D). Lesioned rams were compared to 4 sham and 2 control animals. Blood samples were collected 8 days before, 8 days after, and one month after surgery. Plasma MEL levels were estimated using direct radioimmunoassay. At the end of the experiment, histology and immunohistochemistry of the suprachiasmatic area were performed, and the extent of lesions was evaluated using a computerized image analysis system. Six rams exhibited a complete lesion of the SCN, and in the four remaining animals, the lesions were restricted to the anterior part of the SCN. For all animals, the nocturnal melatonin secretion was altered, but depending on the extent of the lesion, two types of results were observed: Eight days after surgery, in 3 of the 4 rams bearing anterior lesions of the SCN (SCNJ, a nocturnal increase in melatonin secretion still occurred at dusk, but the duration of this secretion extended beyond the end of the night. One month after surgery, melatonin profiles were once again normal, as compared to sham animals. The fourth SCN, ram exhibited a cyclic MEL secretion not synchronized with the light cycle 8 days after surgery, and synchronized with dusk but not dawn one month after surgery. Eight days after surgery, all the rams bearing complete lesions of the SCN (SCNZ) showed strongly altered MEL secretion. This secretion was continuous during the period of sampling, without any characteristic nocturnal peak. Nevertheless, one month after surgery, a nocturnal increase in melatonin secretion synchronized with the dusk was again observed, but the duration of the melatonin secretion was longer than the duration of the night. These results confirm that the SCN mediates the nocturnal pineal gland activity, demonstrate that the anterior part of the sheep SCN is the main drive for the inhibition of the melatonin secretion at dawn and suggest that a positive input to the pineal gland may stimulate MEL secretion at dusk. The partial recovery of the cyclic secretion of melatonin after complete lesioning of the SCN suggests the existence of other structure(s) involved in this rhythmic function.     

Melatonin Inhibits the Activation of Cyclic AMP-Dependent Protein Kinase in Cultured Pars Tuberalis Cells from Ovine Pituitary

The effect of melatonin upon the activation of the intracellular effector enzyme, cyclic AMP (cAMP)-dependent protein kinase (PKA), was investigated in primary cultures of ovine pars tuberalis cells. Incubation of these cells with forskolin caused a rapid and dose-dependent activation of PKA (ED₅₀ 10^～6M). When cells were incubated with forskolin and melatonin simultaneously, the activation of PKA by forskolin was dramatically inhibited. This inhibitory effect of melatonin was dose-dependent (ED₅₀ 10^?10M). Furthermore, treatment with melatonin rapidly deactivated PKA in cells prestimulated with forskolin. When pars tuberalis cell extracts were incubated with 8N₃-[³²P]cAMP, an analogue of cAMP used for photoaffinity labelling of native PKA, specific binding was observed in three bands with M_r of 54, 52 and 48 kd, representing the regulatory subunits of PKA II (in phosphorylated and dephosphorylated forms) and PKA I, respectively. These results indicate that melatonin is a potent inhibitory regulator of cAMP-mediated signal transduction in the ovine pars tuberalis, and suggest that the cellular effects of melatonin in this tissue are mediated by the dephosphorylation of specific substrate proteins.     

Melatonin regulates the synthesis and secretion of several proteins by pars tuberalis cells of the ovine pituitary

The pars tuberalis (PT) of the pituitary may be an important target for melatonin action, but the secretory output of the melatonin-responsive cells is unknown. Using [(35) S]methionine, protein synthesis and secretion have been studied in primary cultures of ovine PT cells, and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Only 4% of the labelled proteins appeared in the medium with the majority retained in the cells. Stimulation of the cells with 10μM forskolin increased the accumulation of several labelled proteins in the medium without corresponding changes in the cell (72, 62, 44, 39, 29, 24, 23, 18 and 14 kd). Two-dimensional gel electrophoresis showed the proteins to have mildly acidic isoelectric points. Melatonin (1 μM) counteracted the stimulatory effect of forskolin on all but one (23 kd) of these secreted proteins. Immunoprecipitation showed this to be prolactin. Furthermore, melatonin alone appeared to have an inhibitory effect on the synthesis and release of proteins into the medium. The synthesis and secretion of the melatonin-responsive proteins was not inhibited by actinomycin D (1 μg/ml), indicating control at the translational level. This contrasts with the regulation of prolactin which is actinomycin D-sensitive. Pulse-chase experiments demonstrated that it requires 30 min for the secretory proteins to appear in the medium, consistent with intracellular processing and packaging prior to secretion. The secretory proteins labelled in the ovine PT, and responsive to melatonin, did not appear to be specific to the PT, as a similar profile of labelled secretory proteins was produced in primary cultures of pars distalis cells. However, melatonin had no effect on the synthesis and secretion of proteins by the pars distalis. These results demonstrate that in the ovine PT melatonin regulates the synthesis and export of several secretory proteins. These are possibly packaging proteins of secretory granules, similar to the granin family of proteins. Thus, the results confirm that melatonin-responsive cells are secretory cells and further imply that the PT-specific product is not a protein.     

The Ovine Pars Tuberalis does not Appear to be Targeted by Melatonin to Modulate Luteinizing Hormone Secretion, but may be Important for Prolactin Release

The pineal hormone, melatonin, transduces photoperiodic information to the neuroendocrine axis of seasonally breeding mammals to regulate reproduction. It is not known where or how melatonin achieves this effect, but the recent identification of the pars tuberalis (PT) as the area with the highest density of melatonin binding sites suggests that this pituitary subdivision may be an important target for the actions of this indoleamine on luteinizing hormone (LH) and prolactin release. The present study was designed to test this hypothesis. Ovariectomized oestradiol-implanted ewes were exposed to inhibitory long days for 85 days and then received melatonin micro-implants (Day 0) in the mediobasal hypothalamus (MBH; n = 7) or PT (Melatonin-PT; n = 5). The effect of these micro-implants was compared to ewes receiving empty micro-implants in the PT (Sham-PT; n = 5). For LH, bi-weekly jugular blood samples were collected and for prolactin, samples were collected every 20 min for 5 h, with the first hour discarded, on Days -4, 26 and 69. Melatonin implanted in the MBH stimulated LH secretion in 3 ewes by Day 46±0 after implantation, and one ewe by Day 67 after implantation. In contrast, no Melatonin-PT or Sham-PT ewes exhibited an increase in LH secretion by the end of the study (Day 70). A subsequent experiment, in which the Sham-PT ewes were implanted with melatonin both subcutaneously and in the PT showed that the micro-implants did not impair the ability of the ovine reproductive neuroendocrine axis to respond to melatonin. For prolactin, both the Melatonin-PT ewes and MBH ewes (which had displayed an increase in LH secretion) exhibited a significant decrease in prolactin secretion by Day 26 and this decrease persisted until the end of the study. This result suggests that melatonin may have two sites of action on prolactin secretion; the MBH and PT, or, if melatonin diffused from the MBH to the PT, or vice versa, that only one of these sites is important. In contrast, these data suggest that the PT is not an important target for the action of melatonin on LH secretion.     

Circuit development in the master clock network of mammals

Daily rhythms are generated by the circadian timekeeping system, which is orchestrated by the master circadian clock in the suprachiasmatic nucleus (SCN) of mammals. Circadian timekeeping is endogenous and does not require exposure to external cues during development. Nevertheless, the circadian system is not fully formed at birth in many mammalian species and it is important to understand how SCN development can affect the function of the circadian system in adulthood. The purpose of the current review is to discuss the ontogeny of cellular and circuit function in the SCN, with a focus on work performed in model rodent species (i.e., mouse, rat, and hamster). Particular emphasis is placed on the spatial and temporal patterns of SCN development that may contribute to the function of the master clock during adulthood. Additional work aimed at decoding the mechanisms that guide circadian development is expected to provide a solid foundation upon which to better understand the sources and factors contributing to aberrant maturation of clock function.