Library

Notes: Abstract: Neuropeptide Y is colocalized with noradrena-line in sympathetic fibers innervating the rat pineal gland. In this article we present a study of the effects and mechanisms of action of neuropeptide Y on the pineal noradrenergic transmission, the main input leading to the rhythmic secretion of melatonin. At the presynaptic level, neuropeptide Y inhibits by 45%, with an EC50 of 50 nM, the potassium-evoked noradrenaline release from pineal nerve endings. This neuropeptide Y inhibition occurs via the activation of pertussis toxin-sensitive G protein-coupled neuropeptide Y-Y2 receptors and is independent from, but additive to, the α2-adrenergic inhibition of noradrenaline release. At the postsynaptic level, neuropeptide Y decreases by a maximum of 35%, with an EC50 of 5 nM, the β-adrenergic induction of cyclic AMP elevation via the activation of neuropeptide Y-Y1 receptors. This moderate neuropeptide Y-induced inhibition of cyclic AMP accumulation, however, has no effect on the melatonin secretion induced by a β-adrenergic stimulation. On the contrary, in the presence of 1 mM ascorbic acid, neuropeptide Y potentiates (up to threefold) the melatonin secretion. In conclusion, this study has demonstrated that neuropeptide Y modulates the noradrenergic transmission in the rat pineal gland at both presynaptic and postsynaptic levels, using different receptor subtypes and transduction pathways.

Notes: : Tryptophan hydroxylase in the rat pineal gland undergoes diurnal rhythmic activity. Rat pineal glands exhibit increased tryptophan hydroxylase activity when incubated with a cyclic AMP analogue in vitro. Cyclic AMP-dependent protein kinase phosphorylates tryptophan hydroxylase, purified from rat brain, without any modification of its enzyme activity under our experimental conditions. Actinomycin O or cydoheximide decreases the stimulating effect of the cyclic AMP analogue on pineal tryptophan hydroxylase activity. Incubation of pineal glands in the presence of [35S]mcthionine showed a cyclic AMP-induced increase in tryptophan hydroxylase synthesis. These results explain the circadian rhythm of tryptophan hydroxylase activity in the rat pineal gland and suggest that the regulation of tryptophan hydroxylase expression by cyclic AMP occurs probably either at the translational level or via transient expression of a transcriptional regulatory element

Notes: The exposure of long day seasonal breeders to a short photoperiod (SP) induces both sexual quiescence and a decrease in pars tuberalis (PT) melatonin receptor density. Therefore, we studied the respective roles of melatonin and testosterone on the regulation of PT melatonin receptors in Syrian hamsters transferred from long photoperiod (LP) to SP. Compared with intact sexually active animals in LP, the density of melatonin receptors was not affected by the absence of melatonin after removal of the pineal gland from animals kept in either SP or LP. In contrast, the presence of a long melatonin peak in the blood which induces gonadal atrophy induced a significant decrease in binding capacity. The SP-induced decrease in PT melatonin receptor density was also observed in castrated animals showing that it was directly regulated by melatonin, independently of circulating testosterone concentrations. However, the absence of testosterone induced an increased binding in LP, while increasing blood testosterone concentration after implantation of one testosterone-filled silastic tube resulted in a decrease in binding both in LP-and in SP-animals. These results indicate that testosterone induces a photoperiod-independent decrease in PT melatonin receptor density. In summary, these results show that both melatonin and testosterone have negative regulatory effects on the density of PT melatonin receptors.

Notes: 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, Bmaax 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 Bmax but not in the Kd, Bmax 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, Bmaax 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.

Notes: Melatonin has been proposed to exert some regulatory actions within the pineal gland itself. The present study examined the effect of melatonin on the release of serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) from rat pineal glands by using an in vitro perifusion system. Melatonin induced a concentration-dependent stimulatory effect on 5-HT secretion from 10−6 M to 10−3 M. Maximal effects were obtained with melatonin 10−3M and concentrations lower than 10−6 M were without effect. The secretion of 5-HIAA was inhibited by melatonin 10−3 and 10−4M, but it was increased when pineals were incubated with 10−5 and 10−6 M of melatonin. The indoleamine secretion was also studied on peripherally denervated rat pineal glands. Basal output of 5-HT from these glands was increased when compared with those from control rats. In contrast, the secretion of 5-HIAA was strongly reduced after removal of the sympathetic input to the pineal gland. Melatonin 10−3 M failed to stimulate 5-HT release from denervated pineal glands, although it inhibited 5-HIAA secretion. In contrast, melatonin 10−5 M enhanced 5-HT release without altering 5-HIAA output. Fluoxetine, a 5-HT uptake inhibitor, produced similar effects than mM concentrations of melatonin on the indoleamine secretion from control pineal glands, but it had no effect on glands taken from peripherally denervated rats. These data suggest that mM concentrations of the pineal hormone are able to stimulate 5-HT release from the pinealocyte, while mM concentrations of melatonin increase extracellular 5-HT by inhibiting its reuptake in the adrenergic nerve endings. These findings are discussed in relation to the possible role of melatonin regulating the intra- and extracellular availability of 5-HT in the pineal gland and its significance as an autocrine factor.

Notes: The mammalian suprachiasmatic nuclei (SCN) contain a circadian clock which is regulated by neuronal photic and non-photic afferences. Among these, the serotonergic input originating from the dorsal raphe nucleus (DRN) is extremely important. In rats, a light pulse administered during the dark period is known to induce the expression of the immediate early gene c-fos and to increase melatonin receptor density in the SCN. The aim of this study was to assess whether, in rats, these two phenomena were regulated by serotonin, acting via 5-HT1A receptors. Three days after pinealectomy, 4 groups of rats were injected i.p. 90 min before sacrifice with respectively: (1) vehicle, (2) the 5-HT1A-agonist 8-OH-DPAT (5 mg/kg), (3) the 5-HT1A-antagonist NAN-190 (10 mg/kg) or (4) NAN-190 and then 8-OH-DPAT. Half of the animals from each group were exposed to light for 60 min before sacrifice and the other half remained in darkness. Sacrifice took place 5 to 6 h after lights off. Our results show that the antagonist NAN-190: (1) completely blocked the photically-induced increase of melatonin receptor density in the SCN, with an IC50=0.352±0.103 mg/kg, and (2) partially blocked (30%) the photic induction of Fos (the protein product of c-fos) in the ventrolateral subdivision of the SCN. The agonist 8-OH-DPAT enhanced the photically-induced increase of melatonin receptors by 10% and decreased the photically-induced increase in Fos by 18%. Both drugs were devoid of any effect in non-light-exposed animals. From these results we may suggest that, in rats, there is a serotonergic control of the neuronal path driving photic information to the SCN. This regulation seems to occur through 5-HT1A or 5-HT1A-like receptors.

Notes: We recently determined that melatonin stimulated serotonin (5-HT) secretion from rat pineal glands by increasing 5-HT release from the pinealocytes (μM melatonin concentrations) and by inhibiting 5-HT uptake in the pineal sympathetic nerve endings (mM melatonin concentrations). The present study investigated whether a single melatonin injection could alter the content of indoleamines in the rat pineal gland, as well as its possible dependence on the daytime of administration. Melatonin (150 μg/kg) was i.p. injected at 8 time points (11.00 h, 14.00 h, 17.00 h, 20.00 h, 23.00 h, 02.00 h, 05.00 h and 08.00 h) to rats kept in 12:12 h light:dark cycle (lights on at 07.00 h). Melatonin injections in the afternoon (17:00 h) and late in the nighttime (02.00 h and 05.00 h) decreased pineal 5-HT content 90 min later. The levels of 5-hydroxyindoleacetic acid (5-HIAA) were also decreased 90 min after the melatonin treatment at 14.00 h, 17.00 h and 02.00 h. The effect of melatonin on 5-HT content was a long-lasting effect (still evident after 180 min) only when injected at 02.00 h, whereas 5-HIAA levels were found to be decreased 180 min after melatonin treatment at 14.00 h and 23.00 h. No changes in these compounds were detected 240 min after melatonin treatment. Moreover, melatonin did not change 5-hydroxytryptophan levels at any of the daytime points studied. By contrast, 90 min after the injection of melatonin at 20.00 h, an increased content of pineal N-acetylserotonin was observed. This effect of melatonin could be mediated through a phase alteration of the pineal N-acetyltransferase activity rhythm by acting on the suprachiasmatic clock, althought a direct melatonin effect on the pineal rhythmic function cannot be excluded. The effects of the hormone on 5-HT and 5-HIAA contents agree with previous findings on the inhibitory effect of pharmacological doses of melatonin on pineal 5-HT uptake, which presumably would result in a decreased intraneuronal content of 5-HT and its acid metabolite. These data point to an acute regulatory action of exogenous melatonin on the pineal melatonin synthesis pathway which seems to be limited to two daytime phases: the afternoon-early evening period and the second half of the night.

Notes: This paper describes the effects of β-adrenergic and peptidergic inputs on serotonin (5-HT) synthesis, outflow and metabolism into melatonin in cultured dissociated rat pinealocytes. The spontaneous outflow of 5-HT from pinealocytes was high as demonstrated by the elevated levels of extracellular 5-HT accumulated in the medium (about 5 ng/h/70,000 pineal cells). The β-adrenergic agonist isoproterenol (ISO) used at concentrations up to 10−6 M induced a moderate (+20–40%) increase in intra- and extracellular 5-HT levels together with a large release of melatonin. At a higher ISO stimulation (10−5 M), the intra- and extracellular levels of 5-HT were significantly (−25–30%) reduced whereas melatonin secretion was dramatically increased. This is interpreted as a large 5-HT mobilization for melatonin synthesis and release, consequently reducing both the intracellular pool and outflow of 5-HT. The peptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide (PACAP) up to 10−7 M induced always a moderate (+20–30%) increase in intra- and extracellular levels of 5-HT. However, the use of nM concentrations of VIP or PACAP together with 10−6 M ISO induced a decrease in 5-HT outflow (−25–30%) and a dramatic increase in melatonin secretion as did 10−5 M ISO alone. Neuropeptide Y (NPY) is another pineal peptide which induced a stimulation of 5-HT outflow (+30–40%) although its effect on melatonin release was marginal. The above results are discussed in term of the multineuronal regulation of the synthetic and secretory activities of the rat pineal gland.

Notes: Temporal organization of the molecular clockwork and behavioral output were investigated in nocturnal rats housed in constant darkness and synchronized to nonphotic cues (daily normocaloric or hypocaloric feeding and melatonin infusion) or light (light–dark cycle and daily 1-h light exposure). Clock gene (Per1, Per2 and Bmal1) and clock-controlled gene (Vasopressin) expression in the suprachiasmatic nuclei was assessed over 24 h. Light and exogenous melatonin synchronized the molecular clock, signaling, respectively, ‘daytime’ and ‘nighttime’, without affecting temporal organization of behavioral output (rest/activity rhythm). By contrast, synchronization to hypocaloric feeding led to a striking temporal change between gene expression in the suprachiasmatic clock and waveform of locomotor activity rhythm, rats then becoming active during the subjective day (diurnal-like temporal organization). When the time of feeding coincided with activity offset, normocaloric feeding also synchronized the locomotor activity rhythm with no apparent switch in temporal organization. Peak of Per2 expression in the piriform cortex occurred between the beginning and the middle of the activity/feeding period, depending on the synchronizer. These data demonstrate that even though the suprachiasmatic clockwork can be synchronized to nonphotic cues, hypocaloric feeding likely acts downstream from clock gene oscillations in the suprachiasmatic nuclei to yield a stable yet opposite organization of the rest/activity cycle.

Notes: The rhythm of melatonin synthesis in the rat pineal gland is under the control of the biological clock, which is located in the suprachiasmatic nucleus of the hypothalamus (SCN). Previous studies demonstrated a daytime inhibitory influence of the SCN on melatonin synthesis, by using γ-aminobutyric acid input to the paraventricular nucleus of the hypothalamus (PVN). Nevertheless, a recent lesion study suggested the presence of a stimulatory clock output in the control of the melatonin rhythm as well. In order to further investigate this output in acute in vivo conditions, we first measured the release of melatonin in the pineal gland before, during and after a temporary shutdown of either SCN or PVN neuronal activity, using multiple microdialysis. For both targets, SCN and PVN, the application of tetrodotoxin by reverse dialysis in the middle of the night decreased melatonin levels. Due to recent evidence of the existence of glutamatergic clock output, we then studied the effect on melatonin release of glutamate antagonist application within the PVN in the middle of the night. Blockade of the glutamatergic input to the PVN significantly decreased melatonin release. These results demonstrate that (i) neuronal activity of both PVN and SCN is necessary to stimulate melatonin synthesis during the dark period and (ii) glutamatergic signalling within the PVN plays an important role in melatonin synthesis.