Diurnal actions of melatonin on epileptic activity in hippocampal slices of rats

Since melatonin receptors have been found in the hippocampus of mammals it has been suggested that melatonin can modulate neuronal functions of hippocampal cells. The effect of melatonin (10 nM/l and 1 microM/l) on frequency and amplitude of epileptiform field potentials (EFP) elicited by low Mg(2+) or by bicuculline was tested in the CA1 region of hippocampal slices of rats. In the low Mg(2+) model, melatonin, applied in a near physiological concentration of 10 nM/l, exerts no effect on EFP in slices prepared at night or during the day. In a concentration of 1 microM/l, however, melatonin enhances the frequency of EFP to approximately 140% in slices prepared during the day. This effect was suppressed through simultaneous administration of the melatonin receptor antagonist luzindole (10 microM/l). In contrast, melatonin did not affect epileptic activity in slices prepared at night. Epileptiform discharges elicited by blocking the GABAergic inhibition (bicuculline model) were not affected by melatonin, either during the day or at night. The results indicate that melatonin affects epileptic activity in a diurnal manner and that the action of melatonin is different in relation to the epilepsy model.     

Melatonin Modulates Thyroid Hormones and Splenocytes Proliferation Through Mediation of Its MT1 and MT2 Receptors in Hyperthyroid Mice

Hyperthyroidism is characterized by an increased metabolic rate with the alteration of immune activity. The pineal hormone melatonin regulates various physiological activities through sensitization of MT1 and MT2 membrane receptors in mammals. In the present study we have evaluated the involvement of MT1 and MT2 receptors in melatonin mediated modulation of thyroid hormones and splenocyte proliferation in experimentally induced hyperthyroidic mice. The l-thyroxine treatment induced the hyperthyroidism in mice evidenced with hypersecretion of T3 and T4 hormones from thyroid gland. Hyperthyroidic state increased the TSH hormone level which might be inducing hyper activity in thyroid gland. Exogenous melatonin suppressed the thyroid hormones level as well as TSH level in circulation. The l-thyroxine treatment increased the splenocyte proliferation and showed synergic effects along with melatonin. l-thyroxine treated mice alone or along with melatonin treatment showed differential expression pattern of MT1 and MT2 receptors protein in thyroid and spleen tissues. It seems that melatonin regulates thyroid hormones and splenocyte proliferation through activation of MT1 and MT2 receptors.     

Melatonin mediates seasonal adjustments in immune function.

In addition to seasonal changes in reproductive function, seasonal changes in immune function are mediated by the pineal hormone, melatonin. Melatonin affects immune function both indirectly, acting through other hormones, and directly by acting on components of the immune system. Melatonin also affects tumorigenesis and tumor development. We hypothesize that many of the indirect effects of melatonin on immune function are mediated through glucocorticoids, and appear to be part of an integrated series of adaptations to manage energy. Direct effects of melatonin on immune function appear to be mediated by melatonin receptors on lymphatic tissue or on immune cells in circulation. Winter is energetically demanding and stressful; thermoregulatory demands typically increase when food availability decreases. Individuals would enjoy a survival advantage if seasonally recurring stressors could be anticipated and countered by bolstering immune function. To summarize, melatonin may be part of an integrative system to coordinate reproductive, immunologic and other physiological processes to cope successfully with energetic stressors during winter.     

Melatonin,immunity and cost of reproductive state in male European starlings.

The effects of reproductive condition and exogenous melatonin on immune function were investigated in castrated European starlings, Sturnus vulgaris. Photorefractory and photostimulated starlings exposed to long days were implanted with melatonin or with blank capsules. Photostimulated starlings with blank capsules exhibited reduced splenocyte proliferation in response to the T-cell mitogen, concanavalin A, compared with the other long-day birds. Exogenous melatonin prevented the suppression of immune function by photostimulation. Photorefractory starlings, with or without melatonin implants, exhibited enhanced immune function compared with photostimulated starlings implanted with blanks. This enhancement was not mediated by endogenous melatonin, but appeared to be related to changes in reproductive state. In addition to the traditional costs of reproduction in birds (e.g. raising of young), there may be a cost of the reproductive state of starlings (i.e. whether they are photorefractory or photostimulated). These data are, we believe, the first to indicate a direct effect of reproductive state on immune function that is independent of both photoperiod (i.e., changes in the duration of melatonin secretion) and gonadal steroids.     

Spontaneous "regression" of enhanced immune function in a photoperiodic rodent Peromyscus maniculatus.

Short days inhibit reproduction and enhance immune function in deer mice (Peromyscus maniculatus). Their reproductive inhibition is sustained by an endogenous timing mechanism: after ca. 20 weeks in short days, reproductive photorefractoriness develops, followed by spontaneous recrudescence of the reproductive system. It is unknown whether analogous seasonal timing mechanisms regulate their immune function or whether enhanced immune function is sustained indefinitely under short days. In order to test this hypothesis, we housed adult male deer mice under long (16 h light day(-1)) or short (8 h light day(-1)) day conditions for 32 weeks or under long day conditions for 20 weeks followed by 12 weeks of short days. Mice under the long day conditions remained photostimulated over the 32 weeks, whereas mice housed under the short day conditions exhibited gonadal regression followed by photorefractoriness and spontaneous recrudescence. Mice transferred to short days at week 20 were reproductively photoregressed at week 32. Total splenocytes, relative splenic mass and mitogen-activated splenocyte proliferation were greater in those mice transferred to short days at week 20 than in those mice housed under either long or short day conditions for 32 consecutive weeks, and immune function in mice exposed to short days for 32 weeks was comparable with that of long day animals. These data suggest that short day enhancement of immune function is not indefinite. With prolonged (< or = 32 weeks) exposure to short days, several measures of immune function exhibit "spontaneous" regression, restoring long day-like immunocompetence. The results suggest that formal similarities and, possibly, common substrates exist among the photoperiodic timekeeping mechanisms that regulate seasonal transitions in reproductive and immune function.     

Immunoregulatory role of melatonin in cancer

Melatonin is a ubiquitous indole amine that plays a fundamental role in the regulation of the biological rhythm. Disrupted circadian rhythm alters the expression of clock genes and deregulates oncogenes, which finally promote tumor development and progression. An evidence supporting this notion is the higher risk of developing malignancies among night shift workers. Circadian secretion of the pineal hormone also synchronizes the immune system via a reciprocal association that exists between the immune system and melatonin. Immune cells are capable of melatonin biosynthesis in addition to the expression of its receptors. Melatonin induces big changes in different immune cell proportions, enhances their viability and improves immune cell metabolism in the tumor microenvironment. These effects might be directly mediated by melatonin receptors or indirectly through alterations in hormonal and cytokine release. Moreover, melatonin induces apoptosis in tumor cells via the intrinsic and extrinsic pathways of apoptosis, while it protectsthe immune cells. In general, melatonin has a profound impact on immune cell trafficking, cytokine production and apoptosis induction in malignant cells. On such a basis, using melatonin and resynchronization of sleep cycle may have potential implications in immune function enhancement against malignancies, which will be the focus of the present paper.     

Effect of melatonin on the oxidative stress in N1E-115 cells is not mediated by mt1 receptors

To explore if protective effect of melatonin on oxidative stress induced by okadaic acid, an inhibitor of protein phosphatases PP1 and PP2A, is mediated by membrane receptors subtype mt1, we used an in vitro model with N1E-115 neuroblastoma cells. We demonstrated that exposure of cells to 50 nM okadaic acid for 2 h induces a reduction in the activity of antioxidative enzymes, and an increase of lipid peroxidation products, while melatonin prevents the effect of okadaic acid. On the other hand, the presence of luzindole, 20 min before adding melatonin, did not cause changes on the effect of the melatonin on oxidative stress. These results seem to indicate that protective effect of melatonin is not mediated by mt1 receptors.     

Melatonin pathway transmits information to terminate pupal diapause in the Chinese oak silkmoth Antheraea pernyi and through reciprocated inhibition of dopamine pathway functions as a photoperiodic counter

The present study investigated the functional involvement of melatonin and dopamine in photoperiodism to terminate pupal diapause in the Chinese oak silkmoth, Antheraea pernyi (Lepidoptera: Saturniidae). Diapause in this long‐day (short‐night) species is maintained during long nights and can be terminated by exposure to a short‐night photoperiod. We observed the effects of melatonin and dopamine and their receptor antagonists on diapause pupae. Melatonin and flupentixol, a dopamine receptor antagonist, terminated pupal diapause even under long‐night photoperiods. Dopamine and luzindole, a melatonin receptor antagonist, retarded adult emergence during short nights, whereas melatonin advanced the timing of adult emergence under the short‐night photoperiod in a manner dependent on the number of injections. The results of the day‐length extension experiment indicated that a change in the photoperiod was immediately detected as mRNA expression of the rate‐limiting enzyme of melatonin production. These findings suggest that the melatonin pathway transmits information on the photoperiod to terminate the pupal diapause of A. pernyi. The melatonin pathway also inhibited the dopamine production system, and the dopamine pathway inhibited the melatonin production system. We propose an insect model of the photoperiodic counter driven by mutual inhibition between the melatonin and dopamine pathways.     

Photoperiod modulates the effects of norepinephrine on lymphocyte proliferation in Siberian hamsters

Siberian hamsters (Phodopus sungorus) rely on photoperiod to coordinate seasonally appropriate changes in physiology, including immune function. Immunity is regulated, in part, by the sympathetic nervous system (SNS), although the precise role of the SNS in regulating photoperiodic changes in immunity remains unspecified. The goal of the present study was to examine the contributions of norepinephrine (NE), the predominant neurotransmitter of the SNS, to photoperiodic changes in lymphocyte proliferation. In experiment 1, animals were maintained in long [16:8-h light-dark cycle (16:8 LD)] or short days (8:16 LD) for 10 wk, and splenic NE content was determined. In experiment 2, in vitro splenocyte proliferation in response to mitogenic stimulation (concanavalin A) was assessed in spleen cell suspensions taken from long- or short-day hamsters in which varying concentrations of NE were added to the cultures. In experiment 3, splenocyte proliferation was examined in the presence of NE and selective alpha- and beta-noradrenergic receptor antagonists (phenoxybenzamine and propranolol, respectively) in vitro. Short-day animals had increased splenic NE content compared with long-day animals. Long-day animals had higher proliferation compared with short-day animals independent of NE. NE (1 microM) further suppressed splenocyte proliferation in short but not long days. Last, NE-induced suppression of proliferation in short-day hamsters was blocked by propranolol but not phenoxybenzamine. The present results suggest that NE plays a role in photoperiodic changes in lymphocyte proliferation. Additionally, the data suggest that the effects of NE on proliferation are specific to activation of beta-adrenergic receptors located on splenic tissue. Collectively, these results provide further support that photoperiodic changes in immunity are influenced by changes in SNS activity.     

Intracellular glucocorticoid receptors in spleen,but not skin,vary seasonally in wild house sparrows (Passer domesticus)

Over the short-term and at physiological doses, acute increases in corticosterone (CORT) titres can enhance immune function. There are predictable seasonal patterns in both circulating CORT and immune function across many animal species, but whether CORT receptor density in immune tissues varies seasonally is currently unknown. Using radioligand binding assays, we examined changes in concentrations of glucocorticoid receptors (GR) and mineralocorticoid receptors (MR) in spleen and skin in wild-caught house sparrows in Massachusetts during six different life-history stages: moult, early winter, late winter, pre-egg-laying, breeding and late breeding. Splenic GR and MR binding were highest during the pre-laying period. This may help animals respond to immune threats through increased lymphocyte proliferation and/or an increase in delayed-type hypersensitivity reactions, both of which CORT can stimulate and in which spleen is involved. A decrease in splenic GR and MR during the late breeding period coincides with low baseline and stress-induced CORT, suggesting immune function in spleen may be relatively CORT-independent during this period. We saw no seasonal patterns in GR or MR in skin, suggesting skin''s response to CORT is modulated primarily via changes in circulating CORT titres and/or via local production of CORT in response to wounding and other noxious stimuli.     

Daily and Seasonal Rhythms in Immune Responses of Splenocytes in the Freshwater Snake,Natrix piscator

Present study was designed to examine daily and seasonal variability in the innate immune responses of splenocytes in the fresh water snake, Natrix piscator. Animals were mildly anesthetized and spleen was aseptically isolated and processed for macrophage phagocytosis, NBT reduction, nitrite production, splenocyte proliferation and serum lysozyme activity. Samples were collected at seven time points, viz., 0000, 0400, 0800, 1200, 1600, 2000 and 0000 h during three different seasons, namely summer, winter and spring. Cosinor analysis revealed that percent phagocytosis had a significant 24-h rhythm during summer and spring seasons. The peaks of rhythms in NBT reduction and nitrite release occurred in the morning hours at 10.88 h and 8.31 h, respectively, in winter. A significant 24-h rhythm was also observed in lysozyme concentration and splenocyte proliferation (both Basal and Concanavalin A stimulated) in all three seasons. A significant phase shift in splenocyte proliferation was obtained with a trend of delayed phase shift from winter to spring and from spring to summer. Of the nine variables, significant annual (seasonal) rhythms were detected in almost all variables, excluding phagocytic and splenosomatic indices. All rhythmic variables, except spleen cellularity, exhibited tightly synchronized peaks coinciding with the progressive and recrudescence phases of annual reproductive cycle. It is concluded that the snake synchronizes its daily and seasonal immune activity with the corresponding external time cues. The enhancement of immune function coinciding with one of its crucial reproductive phases might be helping it to cope with the seasonal stressors, including abundance of pathogens, which would otherwise jeopardize the successful reproduction and eventual survival of the species.     

L-Arginine restores splenocyte functions after trauma and hemorrhage potentially by improving splenic blood flow

Several studiesindicate that immune responses are markedly depressed early after onsetof hemorrhage. Decreased organ blood flow has been implicated in thepathophysiology of altered immune responses after trauma-hemorrhage. Inthis regard, administration ofL-arginine has been shown torestore depressed intestinal and hepatic blood flow aftertrauma-hemorrhage, probably due to provision of substrate forconstitutive nitric oxide synthase (cNOS). It remains unknown, however,whether administration ofL-arginine also amelioratesdepressed splenic blood flow and whether this agent has any salutaryeffects on depressed splenocyte functions after trauma-hemorrhage. Malerats underwent sham operation or laparotomy and were bled to andmaintained at a mean arterial blood pressure of 40 mmHg until 40% ofmaximum shed blood volume (MBV) was returned as Ringer lactate (RL).Hemorrhaged rats were then resuscitated with RL (4 times MBV over 1 h).During resuscitation, rats received 300 mg/kgL-arginine or saline (vehicle)intravenously; 4 h later, splenic blood flow, splenocyte proliferation,and splenocyte interleukin (IL)-2 and IL-3 were determined.Administration of L-arginineimproved depressed splenic blood flow and restored depressed splenocytefunctions after trauma-hemorrhage. Therefore, provision ofL-arginine during resuscitationafter trauma-hemorrhage should be considered a novel and safe approachfor improving splenic organ blood flow and depressed splenocytefunctions under such conditions.

     

Photoperiodic Mediation of Seasonal Breeding and Immune Function In Rodents: A Multi-Factorial Approach

SYNOPSIS. Winter is energetically-demanding; thermoregulatorydemands increase when food availability usually decreases. Physiologicaland behavioral adaptations, including termination of breeding,have evolved among nontropical animals to cope with winter energyshortages. Presumably, selection for mechanisms that permitphysiological and behavioral anticipation of seasonal ambientchanges have led to current seasonal breeding patterns for manypopulations. Energetically—challenging winter conditionscan directly induce death via hypothermia, starvation, or shock;surviving these demanding conditions likely evokes significantstress responses. The stress of coping with energetically-demandingconditions may increase adrenocortical steroid levels to theextent that immune function is compromised. Individuals wouldenjoy a survival advantage if seasonally-recurring stressorscould be anticipated and countered by shunting energy reservesto bolster immune function. The primary environmental cue thatpermits physiological anticipation of season is daily photoperiod,a cue that is mediated by melatonin. However, other environmentalfactors, such as low food availability and ambient temperatures,may interact with photoperiod to affect immune function anddisease processes. Laboratory studies of seasonal changes inmammalian immune function consistently report that immune functionis enhanced in short day lengths. Prolonged melatonin treatmentmimics short days, and also enhances immune function in rodents.In sum, melatonin may be part of an integrative system to coordinatereproductive, immunologic, and other physiological processesto cope successfully with energetic stressors during winter.Social factors influence immune function and changes in socialinteractions may also contribute to seasonal changes in immunefunction. The mechanisms by which social factors are transducedinto immune responses are largely unspecified. In order to understandthe optimization of immune function it is necessary to understandthe interaction of factors, on both mechanistic and functionallevels, that affect immunity.     

Effects of melatonin and antagonists of MT1 and MT2 receptors on somatic pain induced within the fixed circadian rhythm

We studied behavioral pain-related reactions (PRRs) induced in mice by subcutaneous injections of 5% formalin within different phases of the fixed circadian illumination rhythm under conditions of administration of exogenous melatonin and of blocking of MT1 and MT2 melatonin receptors. It was demonstrated that modulation of experimentally induced somatic pain depends considerably on the phase of the preset circadian rhythm. In the norm, the duration of PRRs in the middle of the dark phase was 30% smaller than that in the middle of the light phase. Administration of exogenous melatonin in the middle of the light phase decreased the duration of episodes of noxious behavior by 43%, on average. Injections of melatonin within the dark phase resulted in no significant changes in the duration of PRRs. In the dark phase, the blockade of MT1 receptors by luzindole led to an increase in the duration of PRRs by 45%, as compared with the norm, while in the light phase we observed no significant alterations of this duration under conditions of blocking of the above-mentioned receptors. The blockade of MT2 receptors by prazocine in the middle of dark and light phases increased the durations of PRRs by 92 and 28%, respectively. Our data indicate that the analgesic effect of melatonin depends significantly on the level of this hormone in the organism; in turn, such a level is determined by the illumination conditions. The antinoxious effect of melatonin is mediated by MT receptors, in particular by MT2 receptors. Neirofiziologiya/Neurophysiology, Vol. 39, No. 3, pp. 255–259, May–June, 2007.     

The melatonin receptor antagonist luzindole induces Ca2+ mobilization,reactive oxygen species generation and impairs trypsin secretion in mouse pancreatic acinar cells

BackgroundIn this work we studied the effects of the melatonin receptor-antagonist luzindole (1 μM–50 μM) on isolated mouse pancreatic acinar cells.MethodsChanges in intracellular free-Ca²⁺ concentration, reactive oxygen species production and trypsin secretion were analyzed.ResultsLuzindole induced increases in [Ca²⁺]_i that diminished CCK-8 induced Ca²⁺ mobilization, compared with that observed when CCK-8 was applied alone. Treatment of cells with thapsigargin (1 μM), in the absence of Ca²⁺ in the extracellular medium, evoked a transient increase in [Ca²⁺]_i. The additional incubation of cells with luzindole (10 μM) failed to induce further mobilization of Ca²⁺. In the presence of luzindole a concentration-dependent increase in ROS generation was observed that decreased in the absence of Ca²⁺ or by pretreatment of cells with melatonin (100 μM). Incubation of pancreatic acinar cells with luzindole (10 μM) impaired CCK-8-induced trypsin secretion. Melatonin was unable to revert the effect of luzindole on CCK-8-induced trypsin secretion.ConclusionThe melatonin receptor-inhibitor luzindole induces Ca²⁺-mediated pro-oxidative conditions and impairment of enzyme secretion, which creates a situation in pancreatic acinar cells that might compromise their function.General significanceThe effects of luzindole that we have observed, might be unspecific and could mislead the observations when it is used to study the actions of melatonin on the gland. Another possibility is that melatonin receptors exhibit a basal or agonist-independent activity in pancreatic acinar cells, which might be modulated by melatonin or luzindole.     

Lack of immunological responsiveness to photoperiod in a tropical rodent,Peromyscus aztecus hylocetes

Non-tropical rodents undergo seasonal changes in immune function and disease. It has been hypothesized that seasonal fluctuations in immunity of non-tropical rodents are due to suppressed immune function during harsh winter conditions. A logical extension of this hypothesis is that seasonal changes in immunity should be reduced or absent in tropical rodents that do not experience marked seasonal fluctuations in environmental conditions; however this hypothesis remains to be tested. The present study tested the effects of photoperiod on humoral and cell-mediated immune function of male Aztec mice ( Peromyscus aztecus hylocetes). P. a. hylocetes were housed in long (L:D 16:8) or short days (L:D 8:16) for 10 weeks. Animals were then immunized with the antigen keyhole limpet hemocyanin (KLH). Serum anti-KLH immunoglobulin G (IgG) concentrations and splenocyte proliferation in response to the T-cell mitogen Concanavalin A were assessed. Short-day P. a. hylocetes did not display differences in reproductive or immune measures compared with long-day mice. Collectively, these results suggest that P. a. hylocetes are reproductively and immunologically non-responsive to photoperiod. This lack of immunological responsiveness is likely due to the relative seasonal stability of their environment compared with temperate zone species.     

Immunoprotection in proestrus females following trauma-hemorrhage: the pivotal role of estrogen receptors

Immune responses in proestrus females are not altered after trauma-hemorrhage, whereas they are markedly depressed in males. Elevated levels of female sex steroids appear to be responsible for maintaining immune responses but it remains unknown, whether estrogen per se is responsible. To study this, proestrus female C3H/HeN mice were subjected to laparotomy (i.e., soft tissue trauma) and hemorrhagic shock (35+/-5 mmHg for 90 min, then resuscitated) or sham operation and received the estrogen receptor antagonist EM-800 or vehicle during resuscitation. Two hours following trauma-hemorrhage, splenocyte proliferation, IL-2, IL-3, IFN-gamma release, and splenic macrophage IL-6 release was maintained in vehicle-treated females. In EM-800-treated females, however, these immune parameters were significantly depressed. Following trauma-hemorrhage, Kupffer cell TNF-alpha release and circulating TNF-alpha were increased only in EM-800-treated females. These findings indicate that the ability of proestrus females to maintain immune function following trauma-hemorrhage is estrogen-dependent and mediated via estrogen receptors.     

Immunomodulatory effects of dehydroepiandrosterone in proestrus female mice after trauma-hemorrhage.

Studies indicate that administration of the adrenal steroid dehydroepiandrosterone (DHEA) after trauma-hemorrhage in male mice improved cellular immune functions and reduced mortality rates from subsequent sepsis. There is evidence, however, that DHEA is converted to estrogens in males and that estrogens are immunoprotective after trauma-hemorrhage (TH). In contrast, DHEA in females can be converted to testosterone that has deleterious effects on immune functions. The aim of our study, therefore, was to determine whether administration of DHEA in proestrus females after TH would deteriorate immune responses. Proestrus female C3H/HeN mice (age 7-8 wk) were subjected to laparotomy (i.e., soft tissue trauma induced) and hemorrhagic shock (35 +/- 5 mmHg for 90 min) or sham operation. The mice then received DHEA (100 micro/25 g body wt) or vehicle subcutaneously followed by fluid resuscitation (4x the shed blood volume). Plasma IL-6, splenocyte proliferation, splenocyte IL-2, IL-3, IFN-gamma, IL-10 release, and splenic Mphi IL-1beta, IL-6, IL-10, and IL-12 release were determined 24 h after TH. Plasma IL-6 levels were significantly increased in vehicle-treated females, and DHEA administration markedly attenuated this response. In vehicle-treated females, splenocyte proliferation, IL-2, IL-3, and IFN-gamma release, and splenic Mphi IL-1 beta, IL-6, and IL-12 release were maintained or slightly enhanced after TH. In DHEA-treated females, however, these immune functional parameters were either unaltered compared with vehicle-treated animals or even further enhanced, but surprisingly were not depressed. Moreover, DHEA reduced splenocyte and splenic M phi anti-inflammatory cytokine (i.e., IL-10) production after TH compared with vehicle-treated females. Because DHEA further enhances the immune responsiveness in proestrus females after TH, this hormone might be a useful adjunct even in females for further enhancing immune responses and decreasing the mortality rate after trauma and severe blood loss.