Masayuki Iigo

Demand feeding and locomotor circadian rhythms in the goldfish, Carassius auratus: Dual and independent phasing

In contrast to the common diurnal and nocturnal ways of life, some fish species have been shown to have a dual phasing behaviour. Therefore, the daily pattern of behaviour is not always rigidly confined to the light or dark phase and a diurnal fish may become nocturnal and vice versa. In the present study, the locomotor and feeding activities of single goldfish were simultaneously investigated to examine the existence of such dual behaviour. Nineteen goldfish weighing 97.2 g on average were placed individually in 35-1 glass tanks equipped with an infrared sensor and a newly developed self-feeding device. Fish were exposed to a light:dark (LD) 12:12 h cycle, constant darkness (DD), and 45:45 min LD pulses to study endogenous rhythmicity. Under LD 12:12, the daily pattern of behaviour differed between individual fish; some goldfish were diurnal and others were nocturnal. Furthermore, some of them displayed an extraordinary flexibility in phasing because they were light active but dark feeding, and vice versa. Generally, goldfish tended to be day active, although their feeding habits appeared equally distributed between light and dark phases. Under DD, goldfish showed free-running rhythms that averaged 25.3 +/- 1.8 h and 24.4 +/- 1.7 h for locomotor activity and feeding, respectively, but that were slightly shorter under LD pulses. These results indicate that the type of phasing of locomotor activity did not necessarily decide the feeding phase; much of this is explained by the fact that goldfish were self-fed. Flexibility in phasing and a certain degree of independence between locomotor and feeding activities could be seen as an adaptative response of the highly adaptable circadian system of fish.

Effects of season, temperature, and photoperiod on plasma melatonin rhythms in the goldfish, Carassius auratus

Abstract: Effects of season, environmental temperature, and photoperiod on plasma melatonin concentrations were studied in the goldfish, Carassius auratus. When goldfish were reared under natural conditions, melatonin levels at mid‐dark exhibited seasonal changes, with higher levels obtained in June and September than in December and March. When fish were kept under light: dark (LD) cycle of 12: 12 at 5, 15, or 25°C during March‐April, temperature‐dependent increases in melatonin levels at mid‐dark were observed. When animals were maintained under LD 16: 8 or LD 8: 16 in combination with temperature changes (5, 15, and 25°C) during January‐February, the duration of nocturnal elevation in melatonin was controlled by the length of the scotophase while the amplitude was influenced by environmental temperature. These results indicate that plasma melatonin profiles in the goldfish exhibit seasonal changes that are regulated by both photoperiod and temperature.

Melatonin signal transduction in the goldfish, Carassius auratus.

Generation and reception of melatonin signals in the goldfish, Carassius auratus, are reviewed. The photoreceptive pineal gland of the goldfish generates circulating melatonin rhythms according to a given photoperiod under light-dark cycles and in a circadian manner under continuous dark conditions. Melatonin is also produced in the retina in a similar fashion. Melatonin produced in the pineal gland and retina is considered to act as internal zeitgeber in the brain and retina, respectively, controlling various physiological events via specific melatonin binding sites that are coupled with G protein. The goldfish exhibit clear diurnal locomotor activity rhythms under light-dark cycles and free-running rhythms under constant conditions. However, the relationship between melatonin and locomotor activity rhythms in the goldfish remains unclear. Further studies should be required to demonstrate the roles of melatonin in the circadian system in this species.

Administration of Melatonin and Related Indoles Prevents Exercise-Induced Cellular Oxidative Changes in Rats

In an attempt to define the role of the pineal hormone melatonin and two analogues (5-methoxytryptamine, 5MT, and 6-hydroxymelatonin, 6HM) in limiting oxidative stress, the present study investigated the changes in glutathione, lipid peroxidation, and the activity of the antioxidant enzyme glutathione peroxidase after exercise (swimming for 60 min) with or without treatment with the indolamines mentioned. Lipid peroxidation was measured by estimating tissue levels of malondialdehyde and 4-hydroxyalkenals; the experimental animals in these studies were male Sprague-Dawley rats. In the liver, swimming exercise increased the levels of reduced glutathione (GSH) and also significantly increasing oxidized glutathione (GSSG), while decreasing the GSH/GSSG ratio, an index directly related to oxidative stress. When the animals were treated with melatonin, the concentrations of GSH and GSSG were also increased after swimming; however, no reduction in the GSH/GSSG ratio appeared. In the animals treated with 6HM the changes were the same as in those treated with melatonin. In muscle as well, the concentration of GSH and the GSH/GSSG ratio were decreased following 60 min of swimming. Pretreatment of the rats with melatonin prevented these effects. Pretreatment of the rats with both 5MT and 6HM also prevented the changes. Brain GSH/GSSG ratio was not affected by either exercise or indolamine administration. Swimming enhanced lipid peroxidation in the liver, muscle and brain; however, this was prevented in animals treated with melatonin or 6HM before swimming. Glutathione peroxidase was significantly elevated after exercise in the brain but not in the liver and muscle. It is concluded that swimming imposes a severe oxidative stress and suggests that melatonin and, to a lesser degree, 5MT and 6HM confer protection against the oxidative damage associated with swimming for 60 min. This mechanism may be reasonably attributed to their indole structure, which possibly allows these molecules to act as free-radical scavengers.

Circadian rhythms of locomotor activity in the goldfish Carassius auratus

Locomotor activities in the goldfish Carassius auratus were recorded under light-dark (LD) and constant light regimens. Under LD 12:12, all goldfish adjusted to given LD cycles. Activity patterns were classified into three types: L-type activity during the photophase (83%), LD-type activity both during the photophase and the scotophase (10%), and D-type activity during the scotophase (7%). However, these patterns were not fixed, but flexible. Under constant conditions, goldfish exhibited circadian locomotor activities: The proportions of appearance of circadian rhythms and tau values under constant darkness (DD), constant light (LL, 500 lx) and constant dim light (dimLL, 20 lx) were 57% (24.4 +/- 1.6 h, [mean +/- SE], n = 34), 57% (25.2 +/- 2.5 h, n = 35) and 67% (26.0 +/- 3.4 h, n = 10), respectively. These results indicate that the goldfish exhibit flexibility in phasing of locomotor activity rhythms under LD 12:12. In addition, the coupling between circadian clocks and locomotor activities does not seem strong, although the diel locomotor activity in the goldfish is regulated, in part, by a circadian clock. These results are discussed in context with the structure of the teleostean circadian system.

Characteristics, day-night changes, subcellular distribution and localization of melatonin binding sites in the goldfish brain

Melatonin binding sites in the goldfish brain were characterized by radioreceptor assay using 2-[125I]iodomelatonin as the radioligand. Specific binding of 2-[125I]iodomelatonin was rapid, stable, saturable and reversible. Saturation experiments demonstrated that 2-[125I]iodomelatonin binds to a single class of receptor site with an affinity constant (Kd) of 29.8 +/- 0.7 pM and a total binding capacity (Bmax) of 11.47 +/- 0.33 fmol/mg protein at mid-light. At mid-dark, the Bmax value decreased significantly to 7.90 +/- 0.23 fmol/mg protein (P < 0.01) with no significant variation in the Kd value (33.8 +/- 1.5 pM). Competition experiments revealed the following order of pharmacological affinities: 2-iodomelatonin > melatonin > 6-hydroxymelatonin > N-acetyl-5-hydroxytryptamine > 5-methoxytryptamine > 5-methoxytryptophol > 5-methoxyindole-3-acetic acid. 5-Hydroxytryptamine, 5-hydroxytryptophol, 5-hydroxyindole-3-acetic acid, norepinephrine and acetylcholine exhibited no inhibition. Subcellular distribution of melatonin binding sites was demonstrated to be greatest in the P2 and P3 fractions as compared with the P1 fraction. Localization of melatonin binding sites in discrete brain areas was determined to be highest in the optic tectum-thalamus and hypothalamus, intermediate in the telencephalon, cerebellum and medulla oblongata, and lowest in the olfactory bulbs and pituitary gland. These results suggest that characteristics of melatonin receptors are highly conserved during evolution and that in this species melatonin plays neuromodulatory roles in the central nervous system through specific receptors.

Female-soiled bedding induced Fos immunoreactivity in the ventral part of the premammillary nucleus (PMv) of the male mouse

Previous studies have indicated that the ventral part of the premammillary nucleus (PMv) of rodents is involved in the regulation of aggressive and male mating behavior, although the precise physiological function of the PMv is still unclear. To analyze the physiological role of the PMv in male mating behavior, the effects of exposure to bedding soiled by female mice on Fos immunoreactivity (Fos-ir), an early marker of neuronal activation, were studied in the PMv and some sex-related nuclei. We observed that exposure to female-soiled bedding induced Fos-ir expression in the PMv of the male mouse. Although Fos-ir positive cells were found in the posterodorsal part of the medial amygdaloid nucleus and in the posteromedial cortical amygdaloid nucleus, which are terminals of the neuronal projections from the main and accessory olfactory bulbs, the numbers of Fos-ir cells in those nuclei were not affected by exposure to female-soiled bedding. Moreover, Fos-ir was not detected in the ventromedial hypothalamic nucleus. It is well established that soiled bedding is useful as a source of chemosensory substances, which include pheromones. Thus, our findings, in agreement with previous behavioral and anatomical data, suggest that the PMv plays a role in initiating male copulative behavior that is induced by a female mice pheromone(s).

DAILY CYCLES IN PLASMA AND OCULAR MELATONIN IN DEMAND-FED SEA BASS, DICENTRARCHUS LABRAX L.

Abstract Melatonin is regarded as an internal zeitgeber, involved in the synchronization to light of the daily and seasonal rhythms of vertebrates. To date, plasma and ocular melatonin in fish have been extensively surveyed almost solely in freshwater species – with the exception of some migrating species of salmonids. In the present paper, melatonin levels of a marine species (sea bass, Dicentrarchus labrax L) were examined. In addition, the daily rhythms of the demand-feeding activity of sea bass, a fish species characterized by a dual phasing capacity (i.e. the ability to switch between diurnal and nocturnal behaviour), were investigated before sampling. Sea bass, distributed in 12 groups of four fish and kept under constant water temperature and salinity, were exposed to a 12 h light:12u2009h dark cycle (200:0 lx, lights on at 0800 hours). After 4 weeks recording, the animals were killed at 0900, 1200, 1400, 1600, 1900, 2100, 2400, 0200, 0400, 0700 and 0900 hours. Actograms of demand-feeding records revealed a nocturnal feeding behaviour, with some cases of spontaneous inversions in phasing. Melatonin levels in plasma peaked in the middle of the dark phase, dropping after lights on. Melatonin in the eye, on the contrary, exhibited an inverse profile, with high levels during daytime and low levels at night. These results suggest that melatonin in the plasma and the eye may act independently on the flexible circadian system of sea bass.

Effect of melatonin administration on qPer2, qPer3, and qClock gene expression in the suprachiasmatic nucleus of Japanese quail

Temporal changes of mRNA expression of three clock genes, qPer2, qPer3 and qClock, were studied in the suprachiasmatic nucleus (SCN) of Japanese quail under different light conditions, as well as under the condition of continuous melatonin. In addition, the expression of melatonin receptor genes, Mel1a and Mel1c, in the SCN were also examined. The expression of qPer2 mRNA showed robust oscillation during both light and dark (LD) 12:12 cycles and under constant dark conditions (DD), but did not exhibit circadian rhythmicity in constant light conditions (LL), instead being expressed at a consistently high level. Expression of qPer3 also showed robust oscillation under both LD and DD conditions. Unlike qPer2 however, qPer3 mRNA expression remained rhythmic under LL conditions. Contrary to the findings on the other clock genes, no remarkable rhythmicity was detectable in either light condition. Both Mel1a and Mel1c mRNAs were detected in the SCN, however, Mel1a mRNA levels were higher than Mel1c and showed daily rhythmicity. Although implantation of melatonin tubes caused constant high levels of plasma melatonin and consequently masked the endogenous daily melatonin rhythm, no significant differences in the expression pattern of any of the three clock genes were observed between birds with and without constant melatonin. In addition, a single injection of melatonin did not affect mRNA expression of these clock genes. These results suggest that melatonin does not affect transcription of clock genes, but may act on the mechanism of synchronization among SCN oscillatory cells.

Circadian variations of melatonin binding sites in the goldfish brain.

Daily and circadian variations in melatonin binding sites in the brain of goldfish (Carassius auratus) were examined by radioreceptor assay. Under light-dark cycles of 12:12 h, the density (B(max)) of melatonin binding sites in the brain exhibited daily variations with a peak and a trough seen around the light offset and 2 h before light onset, respectively. The affinity (K(d)) exhibited no variation. The rhythm in the B(max) persisted even under constant darkness with higher levels during the late subjective-day. These results indicate that the density of melatonin binding sites in the goldfish brain is regulated by the circadian clock.