Yossi Anis

Effects of long-term administration of melatonin and a putative antagonist on the ageing rat

Adult rats were treated with either melatonin, the putative melatonin antagonist N-(2,4 dinitrophenyl)-5-methoxytryptamine (ML-23), their combination, or a vehicle for 16 months via the drinking water. The survival rates, serum testosterone and densities of 125I-melatonin binding sites in the medulla-pons and hypothalamus of the animals at the age of 27-29 months were significantly higher in the melatonin than vehicle-treated group. Surprisingly, ML-23 without or with melatonin, also prolonged the life-span of the aged animals. ML-23 treatment greatly increased 125I-melatonin binding in the medulla-pons whereas this increase was prevented by melatonin supplementation. Thus melatonin can attenuate age-related decrease in survival rates, testosterone and brain 125I-melatonin binding sites, while chronic blockade by the putative antagonist also elicits melatonin-mimetic responses, perhaps by effecting supersensitivity.

Diurnal variations in melatonin binding sites in the hamster brain: impact of melatonin☆

The distribution of 125I-melatonin binding sites in the male Syrian hamster brain was recorded at 3 times over a 24 h period. The binding in the hypothalamus, hippocampus, medulla-pons and midbrain of the hamsters varied significantly over the 24 h period with different patterns and phases. No such variations were observed in the parietal cortex. Daily morning (10.00 h) or late afternoon (18.00 h) injections of melatonin for 28 days markedly increased the serum concentrations of melatonin at all times recorded. Serum concentrations of testosterone were significantly lower in animals injected with melatonin in the late afternoon than in the untreated controls; no such decrease was observed in animals injected in the morning despite the continuously elevated levels of circulating melatonin. The daily melatonin injections did not significantly affect 125I-melatonin binding in the hypothalamus, parietal cortex and medulla-pons. In the midbrain, 125I-melatonin binding decreased regardless of the time of injection. In the hippocampus, morning melatonin injections caused a marked decrease in 125I-melatonin binding at all times recorded whereas melatonin injected in the late afternoon led to a decrease in 125I-melatonin binding at 10.00 h only. These results indicate diurnal variations in 125I-melatonin binding sites in discrete brain areas of the golden hamster, persisting despite prolonged duration of elevated levels of circulating melatonin. The differential effects of timed melatonin injections on the hippocampal 125I-melatonin binding sites are positively correlated with the counter-antigonadal response produced by morning melatonin injections.

Impact of circulating testosterone on iodomelatonin binding sites in the male rat brain

The effects of castration and subsequent testosterone and estradiol treatment and of a single injection of ethylene-1,2-dimethanesulphonate (EDS) on the distribution of [2-125I]iodomelatonin ([ 125I]melatonin) binding sites in the male rat brain were investigated. Castration produced a marked testosterone-reversible decrease in [125I]melatonin binding in the male rat brain, particularly in the hypothalamus and hippocampus. In contrast, [125I]melatonin binding in the parietal cortex, medulla-pons and cerebellum was generally unaffected by castration. Estradiol did not reverse the effect of castration on [125I]melatonin binding. A single injection of EDS which causes the destruction of Leydig cells led to a marked decrease in [125I]melatonin binding in the brain of the rats between 3 and 7 days after treatment. This decrease correlated with the decline in serum concentrations of testosterone. Specific [125I]melatonin binding and serum concentrations of testosterone subsequently increased to control levels within 37 days after treatment in accord with the repopulation of Leydig cells. The results clearly show that testosterone regulates the density of melatonin receptors in the hypothalamus and hippocampus of the male rat.

Daily scheduling of the golden spiny mouse under photoperiodic and social cues

A most important function of the circadian system is to ensure that behaviors and metabolism are appropriately timed with respect to the light/dark cycle and photoperiod. Ecological constraints can perturb the daily schedules; would they also impair photoperiodic adaptations? A natural model exists in the golden spiny mouse (Acomys russatus), which is nocturnal, but driven into diurnal activity when sharing the habitat with its congener, A. cahirinus. We show here that the presence of A. cahirinus alters the diurnal rhythms of body temperature and urine volume, delays excretion of the major melatonin metabolite, 6-sulfatoxymelatonin (6-SMT), and increases 2-deoxyglucose uptake by the suprachiasmatic nuclei in A. russatus. Nevertheless, a clear photoperiod effect on urine volume and 6-SMT rhythms was observed. These results indicate that the circadian system can adapt to major changes in daily scheduling without impairing daylength measurement, and consequently seasonal adaptation.

Pinealectomy but not Melatonin Supplementation Affects the Diurnal Variations in 125I-Melatonin Binding Sites in the Rat Brain

The distribution of melatonin binding sites in synaptosomal preparations from five brain areas of sham-operated and pinealectomized young male rats (maintained in a 14 h light: 10 h darkness cycle; lights on at 5.00 h) was recorded at 10.00, 18.00 and 24.00 h, 18 days after surgery, using 125I-melatonin as a probe. The densities of 125I-melatonin binding sites in the medulla-pons, hippocampus and hypothalamus of the pinealectomized rats, exhibited clear diurnal variations. However, the densities of binding sites in these brain areas at 18.00 h were lower in the pinealectomized animals than at the other times of day tested, whereas in the sham-operated controls, the binding at 18.00 h was higher than at the other times of day. No diurnal variations were evident in the midbrain and cerebellum of the pinealectomized animals. The apparent affinities of the binding sites toward the ligand in the various brain areas were similar in the pinealectomized and sham-operated animals and did not significantly vary at any of the times recorded. Oral supplementation of melatonin to the rats via drinking water had no effect on the diurnal variations in 125I-melatonin binding in the pinealectomized rat brain. The results indicate that the diurnal variations in 125I-melatonin binding sites in the rat brain are not generated by the pineal but are affected by removal of the gland.

Castration affects brain iodomelatonin binding in hamsters maintained in long but not short days

The effects of castration on 2-[125I]iodomelatonin ([125I]melatonin) binding sites in discrete brain areas were investigated in male Syrian hamsters exposed to long and short days. In hamsters maintained in long days (14 h light: 10 h darkness), castration produced a marked decrease in [125I]melatonin binding in the brain, particularly in the medulla-pons hypothalamus and hippocampus. Maximal response in the medulla-pons and hypothalamus was observed at 3 days; specific [125I]melatonin binding subsequently increased to reach control levels within 30 days after castration. In the hippocampus, the decrease in [125I]melatonin binding was still evident at 90 days after castration and could be reversed by testosterone. Exposure to short days (8 h light: 16 h darkness) did not affect [125I]melatonin binding in the various brain areas of the intact hamsters; even after 90 days when circulating testosterone decreased to castrated levels, the binding remained as in intact, long-day-housed controls. Moreover, [125I]melatonin binding in the various brain areas of hamsters exposed to short days was unaffected by castration. The results clearly indicate that the regulation by testosterone of melatonin receptors in the medulla-pons, hypothalamus and hippocampus of the male hamster depends on the prevailing photoperiod.

Affinity labeling of melatonin binding sites in the hamster brain

N-Bromoacetyl-2-iodo-5-methoxytryptamine (BIM), a novel derivative of the biologically active melatonin analog, 2-iodomelatonin, was used to identify melatonin binding proteins in synaptosomes from Syrian hamster brain. Incubation of the synaptosomes with BIM resulted in a concentration dependent, irreversible inhibition of 2-125I-iodomelatonin binding. The radioactive form of BIM, N-Bromoacetyl-2-125I-iodo-5-methoxytryptamine (125I-BIM), became covalently attached to three proteins in the synaptosomes, in a concentration dependent manner. These proteins had apparent molecular weight values of 92, 55 and 45 kilodaltons. The incorporation of 125I-BIM into all three proteins was inhibited by BIM greater than 2-iodomelatonin greater than melatonin whereas the melatonin antagonist N-(1,4 dinitrophenyl)- 5-methoxytryptamine (ML-23) selectively inhibited the labeling of the 45 kDa protein. These results indicate that the 92, 55 and 45 KDa polypeptides are melatonin binding proteins.

Involvement of the pineal gland in daily scheduling of the golden spiny mouse

The light-dark cycle is the major time cue for daily and seasonal scheduling of physiological activities. However, non-photic cues (e.g. environmental and social constraints) may also play a significant role. A natural model exists in the golden spiny mouse (Acomys russatus) which is nocturnal when maintained alone but diurnal when sharing a habitat with its congener, the common spiny mouse (A. cahirinus). We have recently observed that the presence of A. cahirinus provokes a major change in the daily rhythms of body temperature (Tb), and urine volume without affecting the melatonin rhythm and photoperiod-induced responses. The apparent lack of interaction between the daily and photoperiodic scheduling was further investigated by studying the significance of the pineal to the modification of A. russatus daily rhythms induced by the presence of A. cahirinus. Lesion of A. russatus pineal gland resulted in diminution of urinary 6-sulfatoxymelatonin (6-SMT) and modification of Tb and urine volume rhythms. However, the modification of Tb and urine volume rhythms provoked by the presence of A. cahirinus were similar in pineal lesioned and sham-operated A. russatus. The non-photic signals released by A. cahirinus did not significantly affect glucose utilization in the suprachiasmatic nucleus of pineal- as well as sham-lesioned A. russatus. Thus, the modification of the daily scheduling of A. russatus by the photoperiod involves the pineal and/or the melatonin rhythm whereas non-photic cues effect a direct (perhaps masking), pineal-independent response to the competitor.

Modification by oxazepam of the diurnal variations in brain125I-melatonin binding sites in sham-operated and pinealectomized rats

Sham-operated and pinealectomized male rats were maintained at 14h light: 10 h dark cycles (lights-on 5.00 h) and injected daily, for 14 days, with oxazepam or vehicle.125I-melatonin binding was recorded in synaptosomes prepared at 10.00, 18.00, and 24.00 h from the hypothalamus, hippocampus and medulla-pons of the rats. In the sham-operated, vehicle treated rats, specific125I-melatonin binding in all brain areas studied was higher at 18.00 h, whereas in the oxazepam-treated animals, binding was higher at 24.00 h than at the other times tested. In the pinealectomized, vehicle-treated rats, the binding recorded at 18.00 h in all three brain areas, was lower than at the other times of day tested. Oxazepam treatment decreased125I-melatonin binding at 24.00 h in the hippocampus and medulla-pons of the pinealectomized rats and did not significantly affect the binding in the hypothalamus. These results indicate the ability of oxazepam, pinealectomy and their combination, to manipulate the diurnal variations in125I-melatonin binding sites in the rat brain.

Melatonin Receptors in Discrete Areas of Rat and Hamster Brain: Modulation by Melatonin, Testosterone and the Photoperiod

There is a distinct light-dark rhythm of melatonin in the blood and other biological fluids of mammals (For review Tamarkin et al., 1985). There is also a pronounced diurnal rhythm in biological response to melatonin (Reiter et al., 1976): Chronic administration of melatonin by multiple late afternoon injections elicits gonadal regression in hamsters (Tamarkin et al., 1976), white-footed mice (Glass and Lynch, 1982) and rats (Reiter et al., 1980) and delays the development of the accessory sex organs in sexually immature rats (Kinson and Peat, 1971; Lang et al., 1983). Melatonin is ineffective in producing the antigonadotrophic effects when injected in the morning (Lang et al., 1984), but in the hamster it prevents the gonadal regression produced by melatonin injected in the late photophase (Chen et al., 1980). It is generally accepted that the brain is the main target of melatonin action in mammals. The responsiveness of the brain to melatonin may critically depend on melatonin receptors. We have therefore studied the circadian variations of melatonin receptors in discrete areas of the rodent brain and their modulation by melatonin.