Dissociation of circadian and light inhibition of melatonin release through forced desynchronization in the rat

Pineal melatonin release exhibits a circadian rhythm with a tight nocturnal pattern. Melatonin synthesis is regulated by the master circadian clock within the hypothalamic suprachiasmatic nucleus (SCN) and is also directly inhibited by light. The SCN is necessary for both circadian regulation and light inhibition of melatonin synthesis and thus it has been difficult to isolate these two regulatory limbs to define the output pathways by which the SCN conveys circadian and light phase information to the pineal. A 22-h light-dark (LD) cycle forced desynchrony protocol leads to the stable dissociation of rhythmic clock gene expression within the ventrolateral SCN (vlSCN) and the dorsomedial SCN (dmSCN). In the present study, we have used this protocol to assess the pattern of melatonin release under forced desynchronization of these SCN subregions. In light of our reported patterns of clock gene expression in the forced desynchronized rat, we propose that the vlSCN oscillator entrains to the 22-h LD cycle whereas the dmSCN shows relative coordination to the light-entrained vlSCN, and that this dual-oscillator configuration accounts for the pattern of melatonin release. We present a simple mathematical model in which the relative coordination of a single oscillator within the dmSCN to a single light-entrained oscillator within the vlSCN faithfully portrays the circadian phase, duration and amplitude of melatonin release under forced desynchronization. Our results underscore the importance of the SCN`s subregional organization to both photic input processing and rhythmic output control.

Ethanol consumption and pineal melatonin daily profile in rats

It is well known that melatonin participates in the regulation of many important physiological functions such as sleep-wakefulness cycle, motor coordination and neural plasticity, and cognition. However, as there are contradictory results regarding the melatonin production diurnal profile under alcohol consumption, the aim of this paper was to study the phenomenology and mechanisms of the putative modifications on the daily profile of melatonin production in rats submitted to chronic alcohol intake. The present results show that rats receiving 10% ethanol in drinking water for 35 days display an altered daily profile of melatonin production, with a phase delay and a reduction in the nocturnal peak. This can be partially explained by a loss of the daily rhythm and the 25% reduction in tryptophan hydroxylase activity and, mainly, by a phase delay in arylalkylamine N-acetyltransferase gene expression and a 70% reduction in its peak activity. Upstream in the melatonin synthesis pathway, the results showed that noradrenergic signaling is impaired as well, with a decrease in beta 1 and alpha 1 adrenergic receptors` mRNA contents and in vitro sustained loss of noradrenergic-stimulated melatonin production by glands from alcohol-treated rats. Together, these results confirm the alterations in the daily melatonin profile of alcoholic rats and suggest the possible mechanisms for the observed melatonin synthesis modification.

Annual pattern of plasma melatonin and progesterone concentrations in hair and wool ewe lambs kept under natural photoperiod at lower latitudes in the southern hemisphere

To study the annual pattern of plasma melatonin and progesterone concentrations in hair [Santa Ines (SI)] and wool [Romney Marsh (RM) and Suffolk (SU)] ewe lambs kept under natural photoperiods at 21 degrees 59'S, 12 ewe lambs (four/breed) were used. For melatonin, blood samples were collected monthly throughout the year at the onset (17:00, 19:00 and 21:00 hr) and end (04:00, 06:00 and 08:00 hr) of the night, and for progesterone the samples were collected in the morning, two to three times a week throughout the year. Plasma melatonin concentrations at different times of the day changed according to the season. In diurnal periods (17:00 and 8:00 hr) no seasonal differences were observed but they became evident in the nocturnal intervals (21:00 and 4:00 hr) and transitional night-day (6:00 hr) times. The patterns of melatonin secretion were higher in winter and autumn than in spring and summer. The patterns of plasma progesterone secretion were affected by interaction between breed and season. There was no seasonal variation in plasma progesterone concentrations for SI females. The progesterone pattern for RM and SU females varied with season. The plasma levels were higher in autumn and winter than in spring and summer. At 21 degrees 59'S hair and wool ewe lambs showed the same annual pattern of plasma melatonin concentration while the annual progesterone profiles were quite different. For SI females this pattern was constant along all seasons and for RM and SU females this pattern was higher during autumn and winter than spring and summer.

Selective protection of the cerebellum against intracerebroventricular LPS is mediated by local melatonin synthesis

Although melatonin is mainly produced by the pineal gland, an increasing number of extra-pineal sites of melatonin synthesis have been described. We previously demonstrated the existence of bidirectional communication between the pineal gland and the immune system that drives a switch in melatonin production from the pineal gland to peripheral organs during the mounting of an innate immune response. In the present study, we show that acute neuroinflammation induced by lipopolysaccharide (LPS) injected directly into the lateral ventricles of adult rats reduces the nocturnal peak of melatonin in the plasma and induces its synthesis in the cerebellum, though not in the cortex or hippocampus. This increase in cerebellar melatonin content requires the activation of nuclear factor kappa B (NF-κB), which positively regulates the expression of the key enzyme for melatonin synthesis, arylalkylamine N-acetyltransferase (AA-NAT). Interestingly, LPS treatment led to neuronal death in the hippocampus and cortex, but not in the cerebellum. This privileged protection of cerebellar cells was abrogated when G-protein-coupled melatonin receptors were blocked by the melatonin antagonist luzindole, suggesting that the local production of melatonin protects cerebellar neurons from LPS toxicity. This is the first demonstration of a switch between pineal and extra-pineal melatonin production in the central nervous system following a neuroinflammatory response. These results have direct implications concerning the differential susceptibility of specific brain areas to neuronal death.

Visual Navigation In The Neotropical Ant Odontomachus Hastatus (formicidae, Ponerinae), A Predominantly Nocturnal, Canopy-dwelling Predator Of The Atlantic Rainforest.

The arboreal ant Odontomachus hastatus nests among roots of epiphytic bromeliads in the sandy forest at Cardoso Island (Brazil). Crepuscular and nocturnal foragers travel up to 8m to search for arthropod prey in the canopy, where silhouettes of leaves and branches potentially provide directional information. We investigated the relevance of visual cues (canopy, horizon patterns) during navigation in O. hastatus. Laboratory experiments using a captive ant colony and a round foraging arena revealed that an artificial canopy pattern above the ants and horizon visual marks are effective orientation cues for homing O. hastatus. On the other hand, foragers that were only given a tridimensional landmark (cylinder) or chemical marks were unable to home correctly. Navigation by visual cues in O. hastatus is in accordance with other diurnal arboreal ants. Nocturnal luminosity (moon, stars) is apparently sufficient to produce contrasting silhouettes from the canopy and surrounding vegetation, thus providing orientation cues. Contrary to the plain floor of the round arena, chemical cues may be important for marking bifurcated arboreal routes. This experimental demonstration of the use of visual cues by a predominantly nocturnal arboreal ant provides important information for comparative studies on the evolution of spatial orientation behavior in ants. This article is part of a Special Issue entitled: Neotropical Behaviour.

Melatonin in maturation media fails to improve oocyte maturation, embryo development rates and DNA damage of bovine embryos

Melatonin (MEL) acts as a powerful scavenger of free radicals and direct gonadal responses to melatonin have been reported in the literature. Few studies, however, have evaluated the effect of MEL during in vitro maturation (IVM) on bovine embryos. This study tested the addition of MEL to maturation medium (MM) with no gonadotropins on nuclear maturation and embryo development rates and the incidence of DNA damage in resulting embryos. Cumulus-oocyte complexes were aspirated from abattoir ovaries and cultured in MM (TCM-199 medium supplemented with 10% fetal calf serum - FCS) at 39ºC and 5% CO2 in air. After 24 hours of culture in MM with 0.5 µg mL-1 FSH and 5.0 µg mL-1 LH; 10-9 M MEL) or 10-9 M MEL, 0.5 µg mL-1 FSH and 5.0 µg mL-1 LH, the oocytes were stained with Hoechst 33342 to evaluate nuclear maturation rate. After in vitro fertilization and embryo culture, development rates were evaluated and the blastocysts were assessed for DNA damage by Comet assay. There was no effect of melatonin added to the MM, alone or in combination with gonadotropins, on nuclear maturation, cleavage and blastocyst rates. These rates ranged between 88% to 90%, 85% to 88% and 42% to 46%, respectively. The extent of DNA damage in embryos was also not affected by MEL supplementation during IVM. The addition of 10-9 M MEL to the MM failed to improve nuclear maturation and embryo development rates and the incidence of DNA damage in resulting embryos, but was able to properly substitute for gonadotropins during IVM.

Behavioral alarm treatment for nocturnal enuresis

PURPOSES: To investigate the efficacy of alarm treatment in a sample of Brazilian children and adolescents with nocturnal enuresis and relate treatment success to age and type of clinical support. MATERIALS AND METHODS: During 32 weeks, 84 children and adolescents received alarm treatment together with weekly psychological support sessions for individual families or groups of 5 to 10 families. RESULTS: 71% of the participants achieved success, defined as 14 consecutive dry nights. The result was similar for children and adolescents and for individual or group support. The time until success was shorter for participants missing fewer support sessions. CONCLUSIONS: Alarm treatment was effective for the present sample, regardless of age or type of support. Missing a higher number of support sessions, which may reflect low motivation for treatment, increased the risk of failure.

Changes in parents' and self-reports of behavioral problems in Brazilian adolescents after behavioral treatment with urine alarm for nocturnal enuresis

PURPOSE: Compare parents' reports of youth problems (PRYP) with adolescent problems self-reports (APSR) pre/post behavioral treatment of nocturnal enuresis (NE) based on the use of a urine alarm. MATERIALS AND METHODS: Adolescents (N = 19) with mono-symptomatic (primary or secondary) nocturnal enuresis group treatment for 40 weeks. Discharge criterion was established as 8 weeks with consecutive dry nights. PRYP and APSR were scored by the Child Behavior Checklist (CBCL) and Youth Self-Report (YSR). RESULTS: Pre-treatment data: 1) Higher number of clinical cases based on parent report than on self-report for Internalizing Problems (IP) (13/19 vs. 4/19), Externalizing Problems (EP) (7/19 vs. 5/19) and Total Problem (TP) (11/19 vs. 5/19); 2) Mean PRYP scores for IP (60.8) and TP (61) were within the deviant range (T score ≥ 60); while mean PRYP scores for EP (57.4) and mean APSR scores (IP = 52.4, EP = 49.5, TP = 52.4) were within the normal range. Difference between PRYP' and APSR' scores was significant. Post treatment data: 1) Discharge for majority of the participants (16/19); 2) Reduction in the number of clinical cases on parental evaluation: 9/19 adolescents remained within clinical range for IP, 2/19 for EP, and 7/19 for TP. 3) All post-treatment mean scores were within the normal range; the difference between pre and post evaluation scores was significant for PRYP. CONCLUSIONS: The behavioral treatment based on the use of urine alarm is effective for adolescents with mono-symptomatic (primary and secondary) nocturnal enuresis. The study favors the hypothesis that enuresis is a cause, not a consequence, of other behavioral problems.

Nyssomyia intermedia (Lutz & Neiva) and Nyssomyia neivai (Pinto) (Diptera, Psychodidae, Phlebotominae) in a sympatric area: seasonal and nocturnal hourly rhythm in black and white modified Shannon traps

Captures with black and white Shannon traps were undertaken to identify aspects of the behavior of the two cryptic and sympatric species implicated as vectors of cutaneous leishmaniasis, Nyssomyia intermedia (Lutz & Neiva, 1912) and Nyssomyia neivai (Pinto, 1926). The traps were installed side by side, monthly, from July 2001 to June 2002, from 18 to 07 hours, in a peridomicile of Iporanga municipality, state of São Paulo, Brazil. A total of 2,142 specimens were captured, Ny. intermedia (47.4%) and Ny. neivai (50.5%). The white trap was more attractive to both sexes of both species. Males of Ny. neivai predominated (70%) over those of Ny. intermedia on the two traps; on the black trap, the females of Ny. intermedia predominated (63.3%) over those of Ny. neivai (36.7%). Seventy percent of the specimens of both species were captured between 18 and 24 h. Females of Ny. intermedia presented the highest peak at 19-20 h, and those of Ny. neivai at 20-21 h. The highest hourly average for females of Ny. intermedia on the black trap occurred in the winter and that for males in the summer. For Ny. neivai, both sexes predominated in the summer. The two species probably transmit the cutaneous leishmaniasis in the area due to their great predominance.

Melatonin reduces LH, 17 beta-estradiol and induces differential regulation of sex steroid receptors in reproductive tissues during rat ovulation

Background: Melatonin is associated with direct or indirect actions upon female reproductive function. However, its effects on sex hormones and steroid receptors during ovulation are not clearly defined. This study aimed to verify whether exposure to long-term melatonin is able to cause reproductive hormonal disturbances as well as their role on sex steroid receptors in the rat ovary, oviduct and uterus during ovulation. Methods: Twenty-four adult Wistar rats, 60 days old (+/-250 g) were randomly divided into two groups. Control group (Co): received 0.9% NaCl 0.3 mL + 95% ethanol 0.04 mL as vehicle; Melatonin-treated group (MEL): received vehicle + melatonin [ 100 mu g/100 g BW/day] both intraperitoneally during 60 days. All animals were euthanized by decapitation during the morning estrus at 4 a. m. Results: Melatonin significantly reduced the plasma levels of LH and 17 beta-estradiol, while urinary 6-sulfatoximelatonin (STM) was increased at the morning estrus. In addition, melatonin promoted differential regulation of the estrogen receptor (ER), progesterone receptor (PR), androgen receptor (AR) and melatonin receptor (MTR) along the reproductive tissues. In ovary, melatonin induced a down-regulation of ER-alpha and PRB levels. Conversely, it was observed that PRA and MT1R were up-regulated. In oviduct, AR and ER-alpha levels were down-regulated, in contrast to high expression of both PRA and PRB. Finally, the ER-beta and PRB levels were down-regulated in uterus tissue and only MT1R was up-regulated. Conclusions: We suggest that melatonin partially suppress the hypothalamus-pituitary-ovarian axis, in addition, it induces differential regulation of sex steroid receptors in the ovary, oviduct and uterus during ovulation.

Melatonin treatment decreases c-fos expression in a headache model induced by capsaicin

The aim of the present work was to analyze c-fos response within the trigeminal nucleus caudalis (TNC) of pinealectomized rats and animals that received intraperitoneal melatonin, after intracisternal infusion of capsaicin, used to induce intracranial trigeminovascular stimulation. Experimental groups consisted of animals that received vehicle solution (saline-ethanol-Tween 80, 8:1:1, diluted 1:50) only (VEI, n = 5); animals that received capsaicin solution (200 nM) only (CAP, n = 6); animals submitted to pinealectomy (PX, n = 5); sham-operated animals (SH, n = 5); animals submitted to pinealectomy followed by capsaicin stimulation (200 nM) after 15 days (PX + CAP, n = 7); and animals that received capsaicin solution (200 nM) and intraperitoneal melatonin (10 mg/kg) (CAP + MEL, n = 5). Control rats, receiving vehicle in the cisterna magna, showed a small number of c-fos-positive cells in the TNC (layer I/II) as well as the sham-operated and pinealectomized rats, when compared to animals stimulated by capsaicin. On the other hand, pinealectomized rats, which received capsaicin, presented the highest number of c-fos-positive cells. Animals receiving capsaicin and melatonin treatment had similar expression of the vehicle group. Our data provide experimental evidence to support the role of melatonin and pineal gland in the pathophysiology of neurovascular headaches.

Suppressive action of melatonin on the TH-2 immune response in rats infected with Trypanosoma cruzi

Control of the acute phase of Trypanosoma cruzi infection is critically dependent on cytokine-mediated macrophage activation to intracellular killing, natural killer (NK) cells, CD4(+) T cells, CD8(+) T cells and B cells. Cell-mediated immunity in T. cruzi infection is also modulated by cytokines, but in addition to parasite-specific responses, autoimmunity can be also triggered. Importantly, cytokines may also play a role in the cell-mediated immunity of infected subjects. Here we studied the role of cytokines in the regulation of innate and adaptive immunity during the acute phase of T. cruzi infection in Wistar rats. Melatonin is an effective regulator of the immune system. Macrophages and T lymphocytes, which have melatonin receptors, are target cells for the immunomodulatory function of melatonin. In this paper melatonin was orally given via two protocols: prior to and concomitant with infection. Both treatments were highly effective against T. cruzi with enhanced action for the concomitant treatment. The data suggest an up-regulation of the TH-1 immune response as all analyzed parameters, interleukin (IL)-4, IL-10, transforming growth factor-beta 1 and splenocyte proliferation, displayed reduced levels as compared with the untreated counterparts. However, the direct effects of melatonin on immune cells have not been fully investigated during T. cruzi infection. We conclude that in light of the current results, melatonin exerted important therapeutic benefits through its immune regulatory effects.

Melatonin enhances pro-inflammatory cytokine levels and protects against Chagas disease

Pro-inflammatory and modulatory cytokines have an essential role in host defense against human and murine Trypanosoma cruzi infection. Control of T. cruzi parasitism during the acute phase of infection is considered to be critically dependent on direct macrophage activation by cytokines. Melatonin has been proposed to regulate the immune system by affecting cytokine production in immunocompetent cells, enhancing the production of several T helper (Th)1 cytokines. The aims of this work were to evaluate in rats, the influences of exogenous melatonin treatment on T. cruzi-infected host`s immune responses. With this in mind, several immunological parameters were analyzed, including tumor necrosis factor-alpha, gamma-interferon, interleukin-12, nitric oxide (NO) and macrophage count. The melatonin therapy was provided in one of two different treatment regimens, that is, either beginning 7 days prior to infection or concomitant with the infection. Both treatments triggered an up-regulation of the immune response, with the concomitant treatment being more effective; in this case all cytokines studied, with exception of NO, displayed enhanced concentrations and there was a higher number of peritoneal macrophages, which displayed reduced concentrations under melatonin therapy. We conclude that melatonin plays a pivotal role in up-regulating the Th1 immune response thus controlling parasite replication.

Enhanced protection by melatonin and meloxicam combination in experimental infection by Trypanosoma cruzi

P>The aim of this study was to evaluate a possible synergism between melatonin and meloxicam in up-regulating the immune response in male Wistar rats infected with Trypanosoma cruzi during immunosuppression phenomenon, which characterizes the acute phase of the Chagas` disease. Male Wistar rats were infected with the Y strain of T. cruzi. Experiments were performed on 7, 14 and 21 days post-infection. Several immunological parameters were evaluated including gamma-interferon (IFN-gamma), interleukin-2 (IL-2), nitric oxide (NO) and prostaglandin E(2) (PGE(2)). The combined treatment with melatonin and meloxicam significantly enhanced the release of IL-2 and INF-gamma into animals` serum, when compared with the infected control groups during the course of infection. Furthermore, the blockade of PGE(2) synthesis and the increased release of NO by macrophage cells from T. cruzi-infected animals contributed to regulate the production of Th1 subset cytokines significantly reducing the parasitaemia in animals treated with the combination of both substances. Therefore, our results suggest that the association of melatonin and meloxicam was more effective in protecting animals against the harmful actions of T. cruzi infection as compared with the treatments of meloxicam or melatonin alone.

Melatonin and dehydroepiandrosterone combination: does this treatment exert a synergistic effect during experimental Trypanosoma cruzi infection?

Previous studies showed that melatonin or dehydroepiandrosterone (DHEA) enhances the immune response against parasitic pathogens. The present study investigated the in vitro activity of melatonin combined with DHEA in a period of 24 hr during the course of in vivo T. cruzi infection. The in vitro activity of melatonin or DHEA alone, as well as together, were tested for the trypomastigote forms (doses ranging from 0.5 to 128 mu m). In vitro, neither melatonin nor DHEA alone had any activity against trypomastigote forms, although when the highest concentration of combined melatonin and DHEA was used, it was active against the trypomastigote forms of the parasite. However, for this concentration, a quite toxicity on peritoneal macrophages was observed. For in vivo evaluation, male Wistar rats were infected with the Y strain of T. cruzi. They were orally treated with 10 mg/kg body weight/day of melatonin and subcutaneously with 40 mg/kg body weight/day of DHEA. Treatment with melatonin, DHEA and the association showed a significant reduction in the number of blood trypomastigotes during the acute phase of infection as compared to untreated animals (P < 0.05). A significant increase in the number of macrophages and nitric oxide (NO) concentrations were observed during the peak of parasitaemia with melatonin alone or combined with DHEA. However, with DHEA alone the highest concentration of NO was observed (P < 0.05). Moreover, DHEA treatment increased TNF-alpha levels during the infection (P < 0.05). These results show that melatonin, DHEA or the combination of both reduces parasitemia during the acute phase of infection. The combined action of both molecules did not exert a synergic action on the host`s ability to fight infection, and it seems that among all treatments DHEA induces a more efficient immune response.