Time-dependent effects of leptin on food intake and locomotor activity in goldfish.

The present study investigates the possible circadian dependence of leptin effects on food intake, locomotor activity, glycemia and plasma cortisol levels in goldfish (Carassius auratus). Fish were maintained under 12L:12D photoperiod and subjected to two different feeding schedules, one group fed during photophase (10:00) and the other one during scotophase (22:00). Leptin or saline were intraperitoneally injected at two different times (10:00 or 22:00), coincident or not with the meal time. To eliminate the entraining effect of the light/dark cycle, goldfish maintained under 24h light (LL) were fed and leptin-injected at 10:00. A reduction in food intake and locomotor activity and an increase in glycemia were found in goldfish fed and leptin-injected at 10:00. No significant changes in circulating cortisol were observed. Those effects were not observed when leptin was administered during the scotophase, regardless the feeding schedule; neither in fish maintained under LL, suggesting that a day/night cycle would be necessary to observe the actions of leptin administered during the photophase. Changes in locomotor activity and glycemia were only observed in goldfish when leptin was injected at daytime, coincident with the feeding schedule, suggesting that these leptin actions could be dependent on the feeding time as zeitgeber. In view of these results it appears that the circadian dependence of leptin actions in goldfish can be determined by the combination of both zeitgebers, light/dark cycle and food. Our results point out the relevance of the administration time when investigating regulatory functions of hormones.

Melatonin receptors in brain areas and ocular tissues of the teleost Tinca tinca: characterization and effect of temperature.

The aim of the present study was to characterize the central melatonin receptors in brain areas and ocular tissues of the teleost Tinca tinca. We investigated the temperature-dependence of 2-iodo-melatonin ([(125)I]Mel) binding in the optic tectum-tegmentum area and the neural retina. The binding of [(125)I]Mel showed a widespread distribution in brain and ocular tissues, with the highest density in the optic tectum-thalamus and the lowest in hindbrain. The [(125)I]Mel affinity was similar in all the studied tissues, and it was on the order of the low pM range. Saturation, kinetic and pharmacological studies showed the presence of a unique MT(1)-like melatonin binding site. In addition, the non-hydrolysable GTP analog, the GTPgammaS, and sodium cations induced a specific binding decrease in the optic tectum and neural retina, suggesting that such melatonin binding sites in the tench are coupled to G protein. Thus, these melatonin binding sites in optic tectum and neural retina fulfil the requirements of a real hormone receptor, the specific binding is rapid, saturable, and reversible, and is inhibited by GTP analogs. Additionally, a clear effect of temperature on such central melatonin receptors was found. Temperature did not modify the B(max) and K(d), but the kinetics of [(125)I]Mel binding resulted in a highly thermosensitive process in both tissues. Both association and dissociation rates (K(+1) and K(-1)) significantly increased with assay temperature (15-30 degrees C), but the K(d) constant (estimated as K(-1)/K(+1)) remained unaltered. In conclusion, this high thermal dependence of the melatonin binding to its receptors in the tench central nervous system supports the conclusion that temperature plays a key role in melatonin signal transduction in fish.

Binding characteristics and daily rhythms of melatonin receptors are distinct in the retina and the brain areas of the European sea bass retina (Dicentrarchus labrax).

Melatonin is synthesized, with a circadian rhythm, in the pineal organ of vertebrates, high levels being produced during the scotophase and low levels during the photophase. The retina also produces melatonin, although in the case of the European sea bass, its secretion pattern appears to be inverted. In the study described here, radioreceptor assay techniques were used to characterize the melatonin binding sites, their regional distribution and their daily variations. Brain and retina membrane preparations were used in all the binding assays and 2-[125I]iodomelatonin ([125I]Mel) as radioligand at 25 degrees C. The specific binding of [125I]Mel was seen to be saturable, reversible, specific and of high affinity. In all the tissues assayed, the power of the ligands to inhibit [125I]Mel binding decreased in the following order: melatonin>>4-P-PDOT>luzindole> or =N-acetylserotonin, which points to the presence of Mel1-like receptors. The inhibition curves of 4-P-PDOT suggested the presence of two different binding sites in the brain areas, but only one type of site of low affinity in the neural retina. No daily variations in [125I]Mel binding capacity (Bmax) or affinity (Kd) were detected in the brain areas, while a clear rhythm in Kd melatonin receptor affinity and Bmax binding capacity was observed in the retina. Kd and Bmax retinal rhythms were out of phase with the lowest Kd and the highest Bmax occurring at scotophase. This result suggests that retinal melatonin is a paracrine factor able to control receptor desensitization during photophase when ocular melatonin is higher in this species.

Neuroprotection by melatonin from glutamate-induced excitotoxicity during development of the cerebellum in the chick embryo.

This work investigated the ability of melatonin to prevent cell damage in the cerebellar cortex of chick embryo caused by glutamate administration. Cell injury was evaluated estimating, at ultrastructural level, the phenomenon of cell death and the synaptogenesis of the Purkinje cells and the cerebellar glomerular synaptic complex. Administration of glutamate during cerebellar development of the chick provokes excitotoxic neuronal degeneration characterized by a phenomenon of neuronal cell death that exhibits essentially the features of a death pattern described as necrosis and the deletion of synaptogenic processes. Our results show that melatonin has a neuroprotective effect against glutamate-induced excitotoxicity. This effect is morphologically revealed by the lack of neural cell death in the embryos treated with melatonin prior to glutamate injection and also by the degree of a synaptogenesis similar to that exhibited by the control group. Likewise, we corroborate the absence of teratological effects of melatonin on chick cerebellar development. Although the possible mechanisms involved in the neuroprotective effect of melatonin are discussed, i.e., direct antioxidant effects, up-regulating endogenous antioxidant defenses, and inhibiting nitric oxide formation activated by glutamate, further studies are required to establish the actual mechanism involved in the neuroprotective effect of melatonin.

Neuropeptide Y has a stimulatory action on feeding behavior in goldfish (Carassius auratus).

The purpose of the present study was to elucidate the possible role of neuropeptide Y (NPY) in the feeding regulation in fish. We examined the effects of intracerebroventricular (i.c.v.) or intraperitoneal (i.p.) neuropeptide Y administration on food intake in satiated goldfish, at different time intervals postinjection (0-2, 2-8 and 0-8 h). Food intake was significantly increased by i.c.v. administered neuropeptide Y (1 microg) at 2 h postinjection, while no significant differences in food intake were observed after i.p. treatment. The neuropeptide Y receptor antagonist, neuropeptide Y-(27-36), totally counteracted the stimulatory action of neuropeptide Y on feeding. The possible involvement of neuropeptide Y in the eating behavior evoked by food deprivation has been investigated. Food deprivation by either 24 or 72 h significantly increased feeding, and the neuropeptide Y receptor antagonist attenuated such feeding stimulation. From our findings, we suggest, first, that neuropeptide Y is involved in feeding central regulation in goldfish, acting via specific neuropeptide Y receptors, and second, that hypothalamic neuropeptide Y would be released in response to food deprivation, contributing to generate the consequent eating behavior stimulation in Carassius auratus.