T. D. Siopes

Melatonin Rhythms in Quail: Regulation by Photoperiod and Circadian Pacemakers

The profile of melatonin in the eyes, pineal, and blood of Japanese quail was assessed in birds held under LD 16:8 and LD 6: 18 photoperiods. Melatonin levels in all three tissues showed a robust daily rhythm with higher levels occurring at night. The amplitude of the rhythm was depressed and its duration lengthened on LD 6: 18 relative to LD 16:8. The blood melatonin rhythm precisely reflected the rhythms shown by the pineal and eyes, supporting the idea that the blood rhythm is a result of melatonin secretion by both the eyes and pineal.

Does a Biological Clock Reside in the Eye of Quail

The site (intra- vs. extraocular) of the circadian clock driving an ocular melatonin rhythm in Japanese quail was investigated by alternately covering the left and right eyes of individual quail, otherwise held in constant light (LL), for 12-hr periods. This procedure exposed each eye to a light-dark (LD) 12:12 light cycle 180° (12 hr) out of phase with the LD 12:12 light cycle experienced by the other eye. This protocol entrained the melatonin rhythm in the left eye of quail 180° out of phase with the rhythm expressed in the right eye. These results are compatible with the hypothesis that an independent light-entrainable circadian pacemaker resides in each eye; they are incompatible with the hypothesis that a single (or functionally single) extraocular pacemaker drives the ocular melatonin rhythm in both eyes. However, the results are also compatible with a model in which two independent extraocular circadian pacemakers, each with an exclusive photic input from one eye, drive the ocular melatonin rhythm.

Melatonin enhances cellular and humoral immune responses in the Japanese quail (Coturnix coturnix japonica) via an opiatergic mechanism

It is known that melatonin has important immunomodulatory properties in the Japanese quail. However, the mechanism of melatonin action on the immune system is not clearly understood in avian species. In mammals, the immunostimulatory properties of melatonin are mediated by the release of opioid peptides from activated T-lymphocytes. The present study was performed to determine if these same melatonin-induced opioids (MIO) are involved with the immunoenhancing effects of melatonin in quail. Three treatment groups were given melatonin (50 microg/ml) in the drinking water ad libitum along with naltrexone, a known opioid receptor-blocking agent. Melatonin was administered throughout the 3 week study and each bird received a daily intramuscular injection of naltrexone at a dose of 0.1, 1.0, or 10.0 mg/kg. In addition, three control groups were established that received only melatonin, naltrexone, or diluent. Evaluation of the cellular and humoral immune responses was initiated after 2 weeks of treatments. A cutaneous basophil hypersensitivity reaction to phytohemagglutinin (PHA-P) was measured to evaluate the cellular immune response. To evaluate the humoral immune response, primary antibody titers were determined 7 days post-intravenous injection with a Chukar red blood cell (CRBC) suspension. Both the cellular and humoral immune responses were significantly increased by 22 and 34%, respectively, upon melatonin exposure as compared to quail receiving diluent only. Concomitant administration of naltrexone and melatonin significantly reduced the immunoenhancing effect of melatonin across all naltrexone doses. We conclude that melatonin enhances a cellular and humoral immune response in Japanese quail via an opiatergic mechanism.

Pineal melatonin secretion, but not ocular melatonin secretion, is sufficient to maintain normal immune responses in Japanese quail (Coturnix coturnix japonica)

Reports that plasma melatonin is an important immune regulator in avian species have been rather sparse and contradictory. Also, the primary source of immune-modulating melatonin has yet to be determined in birds. In Japanese quail (Coturnix coturnix japonica), the pineal gland and eyes contribute roughly two thirds and one third of the melatonin found in the blood, respectively. Two experiments were conducted to evaluate melatonin as an immune modulator in Japanese quail and to determine the primary source of immune-modulating melatonin in this species. Experiment 1 was designed to evaluate the involvement of the pineal gland and the eyes in immunocompetence. Each of three groups of quail was assigned a surgical treatment and the cellular and humoral immune responses were determined 8 weeks following surgery. The surgical treatments were pinealectomy (Px), sham pinealectomy (SH-Px), and ocular enucleation (eye removal (Ex)). Experiment 2 utilized exogenous melatonin as a replacement to reconstitute immune responses in surgically immunocompromised birds. In this experiment, 50.0 microg/ml of melatonin, or diluent only, was provided to Px and SH-Px birds in the drinking water ad libitum. The cellular and humoral immune responses were determined after 8 weeks of melatonin treatment. In both experiments, a cutaneous basophil hypersensitivity reaction to phytohemagglutinin was measured to evaluate the cellular immune response. To evaluate the humoral immune response, primary antibody titers were determined 7 days postintravenous injection with a Chukar red blood cell suspension. Flow cytometric analysis of peripheral blood lymphocytes was performed to determine the relative percentage of CD4(+) and CD8(+) T- and B-lymphocytes in all treatments of Experiment 2. In Experiment 1, both the SH-Px and Ex surgical treatments produced similar cellular and humoral immune responses, and these responses were significantly greater than those in Px-treated birds. Pinealectomy significantly reduced the cellular and humoral immune responses from SH-Px by 25.8% and 41.3%, respectively. In Experiment 2, Px again resulted in depressed cellular and humoral immune responses. In addition, Px significantly reduced CD8(+) T-lymphocyte numbers compared to SH-Px, while B-lymphocytes remained unchanged. Melatonin administration to Px birds increased the cellular (32.9%) and humoral (30.6%) immune responses to the level of control (SH-Px) birds, although this reconstitution was not due to increased CD8(+) T- or B-lymphocytes. From these data, it was clear that removal of the pineal gland, but not the eyes, reduced cellular and humoral immune responses, which were reconstituted to normal levels by exogenous melatonin. These data suggest that immunodepression is only observed in birds with two thirds of the plasma melatonin removed by pinealectomy. Removal of one third of the plasma melatonin (by ocular enucleation) is not sufficient to reduce cellular and humoral responses in the Japanese quail.

Melatonin can produce immunoenhancement in Japanese quail (Coturnix coturnix japonica) without prior immunosuppression

In recent years, it has been determined that melatonin has important immunostimulatory properties in mammalian and avian species. Typically, this immunoenhancement has only been examined in immunosuppressed animals. The effect of melatonin on normal (unsuppressed) immune systems is yet to be evaluated in avian species. An experiment was performed to determine if transient and/or continuous melatonin treatments could enhance immune functions in Japanese quail without prior immunosuppression. All quail were kept on a short photoperiod (8:16LD) throughout the study. In this experiment, 50.0 microg/ml melatonin was provided ad libitum to adult Japanese quail in the drinking water either continuously or for 3h per day. Control birds received diluent continuously throughout the experiment. Both the cellular and humoral immune responses were determined immediately after 3 weeks treatment. A cutaneous basophil hypersensitivity reaction to phytohemagglutinin (PHA-P) was measured to evaluate the cellular immune response. To evaluate the humoral immune response, primary antibody titers were calculated 7 days post-intravenous injection with a Chukar red blood cell (CRBC) suspension. The cellular and humoral immune responses were significantly elevated in the transient (3h) and continuous (24h) melatonin treatment groups as compared to the control group (0 h). As compared to the control group, the cellular immune response was increased 25% and 38% for the 3 and 24h melatonin treatments, respectively. The humoral immune response was increased 26% and 32% for the 3 and 24h melatonin treatments, respectively. Furthermore, continuous (24h) melatonin availability significantly increased the cellular, but not humoral immune responses as compared to the transient (3h) group, given melatonin for 3h prior to the scotophase (13:00-16:00 h). From these data, it was clear that transient and continuous administration of melatonin increased the cellular and humoral immune responses of Japanese quail without prior immunosuppression. These data suggest that the immunoenhancing effect of melatonin is not limited to reconstitution of weakened immune systems, but can be observed in normal, immunologically unsuppressed birds.

Photostimulated fos-like immunoreactivity in tuberal hypothalamus of photosensitive vs. photorefractory turkey hens.

Photorefractoriness in commercial turkey hens can be viewed as a failure of previously sexually stimulatory photoperiods to maintain egg production via activation of cGnRH I neurons, but the neural locus of photorefractoriness, i.e., where in the brain failure occurs, is not known. We used a c-fos antiserum that detects c-Fos and Fos-related antigens to characterize Fos-like immunoreactivity (FLI) as a measure of neuronal activation. FLI was measured in somatically mature, photosensitive hens (held on short photoperiods [8L:16D] for at least 10 weeks) before (non-photostimulated-photosensitive group) and after 48 h of exposure to long photoperiods (16L:8D; photostimulated-photosensitive group). We also measured FLI in hens that had become photorefractory, transferred to short photoperiods for 1 week--an insufficient time period to reverse photorefractoriness--and then exposed to long photoperiods for 48 h (photostimulated-photorefractory group). FLI was nearly absent in the tuberal hypothalamus of non-photostimulated-photosensitive hens but FLI was abundant in photostimulated-photosensitive hens. FLI was greatly reduced (P<0.01) in the rostral tuberal hypothalamus of photostimulated-photorefractory hens. All hens showed variable extra-tuberal FLI in locations associated with stress, e.g., paraventricular nucleus, lateral septal area, and nucleus taenia. Double-label fluorescence immunohistochemistry with c-fos antiserum and anti-Neu-N, a neuron-specific protein, showed that a substantial fraction of tuberal FLI-positive cells in photostimulated-photosensitive hens were neuronal. These results implicate neurons in the rostral tuberal hypothalamus as a potential neural locus of photorefractoriness, as FLI in this region appears coupled with cGnRH I activation in photostimulated-photosensitive but not photostimulated-photorefractory turkey hens.

Pineal and ocular influences on male and female turkeys: Plasma luteinizing hormone and prolactin levels during gonadal development

Plasma levels of prolactin (PRL) and luteinizing hormone (LH) were determined in pinealectomized (PX) and/or bilateral ocular enucleated (EX) turkey hens and toms during gonadal development. Measurements were obtained weekly for 6 weeks following photo-induced ovarian recrudescence in adult hens and biweekly from 12 to 34 weeks of age in sexually developing toms. Both hens and toms were maintained on a photoperiod of 16L:8D. Plasma PRL levels were significantly correlated with time (P less than 0.01) and were linear with significant slopes (P less than 0.01) in all treatment groups of hens and toms. However, the regression coefficients were positive for the hens and negative for the toms. Both PX and EX reduced PRL levels in the hens. PX did not significantly affect PRL levels of toms but EX depressed PRL levels. In hens LH was not correlated with weeks of photostimulation but in toms LH was positively correlated to age and linear with significant slopes in all treatment groups. There were no significant treatment effects on plasma LH levels of hens or toms. It was concluded that neither the pineal gland nor the eyes affect plasma LH levels of male turkeys during testicular development or female turkeys during photostimulated ovarian recrudescence. Although the eyes affected plasma PRL of both males and females during gonadal development, the pineal only influenced female PRL levels.

Circadian Ovulatory Rhythms in Japanese Quail: Role of Ocular and Extraocular Pacemakers

Previous studies have shown that the circadian system of Japanese quail is composed of multiple photic inputs and multiple oscillators. Among these are extraretinal photoreceptors that mediate both circadian and photoperiodic responses and circadian pacemakers in the eyes that, via neural and hormonal outputs, help to maintain rhythmicity of central circadian clocks (presumably located in the suprachiasmatic area of the hypothalamus). Furthermore, a component of the central circadian system is influenced by reproductive hormones. Under certain conditions, the circadian system of female quail can be induced to split into two circadian components: one driven by ocular pacemakers and one driven by feedback from reproductive hormones. Importantly, ovulation is either inhibited or permitted as these two oscillators (or sets of oscillators) constantly change internal phase relationships with each other, suggesting an “internal coincidence” mechanism in the control of ovulation. The oviposition patterns of quail in light-dark (LD) cycles also support an internal coincidence mechanism. The authors tested the hypothesis that the ocular pacemakers are an important component of an internal coincidence mechanism controlling ovulation by examining the effects of blinding by complete eye removal (EX), and the effects of eye-patching, on the body temperature and oviposition patterns of quail exposed to 24-h LD cycles. They also examined the effects of EX on quail exposed to continuous light (LL) and to continuous darkness (DD). Neither EX nor eyepatching affected the oviposition patterns of birds in LD. Furthermore, robust body temperature and oviposition rhythms continued in EX birds in LL, but body temperature became arrhythmic in DD with the cessation of ovulation. The results do not show a role for ocular pacemakers in the control of ovulation, but they do support the hypotheses that (1) entrainment of the central oscillators by extraretinally perceived light is sufficient to preserve a normal ovulatory pattern in LD in the absence of the ocular pacemakers, and (2) in LL, feedback of reproductive hormones onto the central oscillators is sufficient to organize the circadian system even in the absence of the ocular pacemakers. Whether or not the ocular pacemakers are normally involved in the control of ovulation is still an open question.

Plasma prolactin and luteinizing hormone levels of pinealectomized and enucleated turkey hens

Plasma levels of LH and prolactin were determined in pinealectomized (PX) and/or bilateral ocular enucleated (EX) adult turkey hens maintained in either constant (24LL) or diurnal (16L:8D) lighting. Blood samples were obtained at the following times over a 24-hr period: onset, middle, and end of light period, middle and end of dark period. In experiment 1, the effects of PX alone were evaluated in photorefractory hens exposed to two different photoperiods (24LL and 16L:8D). In experiment 2, the effects of PX and EX were determined in egg-laying and photorefractory hens exposed to a 16L:8D daily photoperiod. Neither PX nor EX significantly altered LH or prolactin levels of egg laying or photorefractory hens at any of the sampling times of the day in either experiment. In experiment 1, LH levels were very similar in both 16L:8D and 24LL but prolactin levels were significantly lower in 24LL than 16L:8D. It was concluded that the pineal gland does not play an essential role in maintaining circulating LH and prolactin levels in constant light or a photoperiod of 16L:8D.

Diurnal variation in the cellular and humoral immune responses of Japanese quail: role of melatonin.

Experiments were conducted to determine if diurnal variations occur in the cellular and humoral immune responses of sexually mature, male Japanese quail and if this diurnal variation is mediated by the daily rhythm of melatonin. In Experiment 1, quail were exposed to LD 12:12 light-dark cycles and immune responses were measured in response to a single antigenic challenge given to different groups every 4h over a 24h period. Diurnal changes occurred in both the cellular and humoral immune responses. The cellular response was higher during the light phase than during the dark phase whereas the opposite was true for the humoral immune response. Experiment 2 was designed to determine if melatonin mediated these diurnal immune responses. Quail were maintained in continuous light (LL) to suppress endogenous melatonin production and half of them were given melatonin in the drinking water for 12h each day for 2 weeks. Contrary to control quail, significant daily variations occurred in both the humoral and cellular immune responses of birds given melatonin. As in Experiment 1, the cellular and humoral immune responses were out of phase with one another, with the humoral response being maximal when melatonin was present. We may conclude that there exists a melatonin dependent diurnal variation in both cellular and humoral immune responses of quail. The responses were inverse to one another during the daily light-dark cycle with the cellular response being maximal during the daily light period and the humoral response being maximal during the daily dark period.