Interstrain differences in activity pattern, pineal function, and SCN melatonin receptor density of rats.

We investigated the possibility that strain-dependent differences in the diurnal pattern of wheel running activity rhythms are also reflected in the melatonin profiles. The inbred rat strains ACI/Ztm, BH/Ztm, and LEW/Ztm. LEW were examined for diurnal [12:12-h light-dark (LD)] wheel running activity, urinary 6-sulphatoxymelatonin (aMT6s) excretion, melatonin concentrations of plasma and pineal glands, and melatonin receptor density in the suprachiasmatic nuclei (SCN). ACI rats displayed unimodal activity patterns with a high level of activity, whereas BH and LEW rats showed multimodal activity patterns with ultradian components and reduced activity levels. In contrast, the individual daily profiles of aMT6s excretion and mean melatonin synthesis followed a unimodal time pattern in all three strains, suggesting that different output pathways of the SCN are responsible for the temporal organization of locomotor activity and pineal melatonin synthesis. In addition, melatonin synthesis at night and SCN melatonin receptor density at day were significantly higher in BH and LEW rats than in ACI rats. These results support the hypothesis of a long-term stimulating effect of melatonin on its own receptor density in the SCN.

Creatinine is an appropriate reference for urinary sulphatoxymelatonin of laboratory animals and humans.

In our studies on diurnal 6-sulphatoxymelatonin (aMT6s) rhythms in various species, we have sometimes obtained fluctuating patterns. In most of these, the volume of individual urine fractions was not accurately measured because of methodological problems. Here, we report a simple method to overcome these problems by using urinary creatinine to estimate urine volume. The benefit of this method is demonstrated in two representative examples of the diurnal aMT6s rhythms of rats, domestic pigs and humans. Because the human urine fractions were collected accurately, the qualitative pattern of the aMT6s rhythm was not altered by using urinary creatinine as a substitute for urine volume. The total creatinine excretion (urine volume x creatinine concentration) was constant within a small range and showed no diurnal rhythm. In rats and pigs, the highly variable aMT6s concentrations relative to urine volume throughout the 24-hr period were changed drastically by referring to creatinine. All aMT6s patterns became stable and qualitatively similar to those of the rest of the group. From these results it can be concluded that creatinine is an adequate substitute for urine volume and a beneficial parameter with which to overcome technical problems with urine collection from laboratory animals or unknown urine volumes in human studies.

6-Sulphatoxymelatonin secretion in different locomotor activity types of the blind mole rat Spalax ehrenbergi.

6-Sulphatoxymelatonin (aMT6S) excretion was examined in the urine of rhythmic and arrhythmic blind subterranean mole rats (Spalax ehrenbergi) to test the correlation between melatonin secretion (as represented by aMT6S) and variability in circadian locomotor activity. Activity pattern was tested in four males, first for a week under short photoperiod [light:dark (LD) 10:14], followed by 10 days in constant darkness (DD). After several months the experiment was repeated under long photoperiod (LD 14:10), followed by DD conditions. Under LD conditions all animals exhibited aMT6S excretion during the dark phase, with a decline just before the onset of light. No correlation was found between activity pattern and melatonin secretion. The animal with the highest melatonin secretion both under LD and DD had an arrhythmic locomotor pattern. The results suggest that in mole rats melatonin secretion and circadian locomotor activity are controlled by two different mechanisms. There were large differences in the aMT6S levels among individuals, suggesting the importance of duration of melatonin secretion over amplitude for gonadal development and thermoregulatory changes. During summer, i.e., before the breeding season, the animals keep a more stable aMT6S secretion than in winter, and the amplitude of secretion is higher under DD vs. LD conditions.

A short red light pulse during dark phase of LD-cycle perturbs the hamster's circadian clock.

In this study we investigated the influence of red light< which naturally occurs during dawn and dusk, on locomotor activity and body temperature rhythms of Djungarian hamsters (Phodopus sungarus). A single weak red light pulse given 2 h before regular lights on had acute as well as long-term effects persisting for several days following exposure. The hamsters immediately stopped their locomotor activity, accompanied by a drop in body temperature. In the following undisturbed nights (ID 16:8) the nocturnal activity stopped earlier than usual. This lasting effect of the light pulse was more pronounced than the acute effect. The activity phase compressed gradually during 3 to 5 days after the light pulse was administered while time of activity onset was almost unaffected. It took 6 to 11 days for complete recovery of the original activity phase. The maximal activity compression and the recovery period depended on the duration of the single red light pulse and its intensity. Red light pulses of 15 min duration were about twice effective as 1 min pulses; and the effect of a red light pulse of 130 mW/m2 was about 1.5 times stronger than a 30 mW/m2 red light pulse. The maximal value of activity phase compression reached in this experiment was 2.5 + 0.2 h with a recovery period of 11.1 +/- 0.3 days following a given red light pulse of 90 mW/m2 and 15 min. The "morning oscillator" seems to be persistently affected. This indicates a very high photosensitivity of the Djungarian hamster's circadian system to red light.

Melatonin synthesis in the retina and pineal gland of Djungarian hamsters at different times of the year.

Daily profiles of melatonin content in the retina of Djungarian hamsters were measured throughout the course of a year. Peak levels of retinal melatonin were found at various times of the day at different seasons. In a subsequent study we determined concurrently daily profiles of nine indole metabolites (tryptophan, 5-hydroxytryptophan, 5-hydroxytryptamin, 5-hydroxyindoleacetic acid, 5-hydroxytryptophol, N-acetylserotonin, melatonin, 5-methoxyindoleacetic acid and 5-methoxytryptophol) in samples of hamster retina and pineal gland at the four different seasons. Measurements were made using HPLC coupled to a coulometric electrode detection system. Large seasonal variations in the levels and daily rhythm amplitudes of the different indole metabolites were found in the pineal glands. Due to technical problems and unknown interfering substances only tryptophan and serotonin could be determined in the retinas at all four seasons. Despite this, the ability to measure levels of several metabolites in the same sample and in different tissues of the same animals permitted more accurate comparisons of the components involved in melatonin synthesis. Our data suggest that the level of retinal melatonin is regulated and controlled locally within the retina itself. However, long-term feedback mechanisms from other sources of melatonin are not excluded but are rather likely.

Urinary 6-sulphatoxymelatonin excretion reflects pineal melatonin secretion in the Djungarian hamster (Phodopus sungorus).

To monitor pineal function in the Djungarian hamster (Phodopus sungorus), we measured the urinary excretion of the melatonin metabolite 6-sulphatoxymelatonin (aMT6s) at 3-hr intervals by radioimmunoassay. Hamsters maintained in either long photoperiod (LP, LD 16:8) or short photoperiod (SP, LD 8:16) showed marked daily rhythms in aMT6s excretion, with elevated levels during the dark phase. In both photoperiods, we found large interindividual differences, mainly in the amplitude of the signal. However, the amplitude as well as the duration of nocturnal aMT6s excretion was higher in SP than in LP. Light exposure at night (180 mW/m2, 30 min) caused a decrease in aMT6s excretion, indicating that the pineal gland is the major source of urinary aMT6s. Moreover, there was a significant correlation between nocturnal pineal/plasma melatonin contents and 24-hr aMT6s excretion. We conclude that, measurements of aMT6s provide a valid and quantitative index of pineal melatonin synthesis in this hamster species. As an advantage in determining pineal melatonin contents, this approach will allow noninvasive long-term studies of individual animals under varying environmental conditions.