Damped oscillation of the lateral hypothalamic multineuronal activity synchronized to daily feeding schedules in rats with suprachiasmatic nucleus lesions.

In male Wistar rats with chronically implanted electrodes, multiple-unit activity (MUA) was recorded from the lateral hypothalamus (LH) and ventromedial hypothalamus (VMH). Blinded rats with bilateral suprachiasmatic nucleus (SCN) lesions showed no circadian rhythm in MUA or motor activity when food was available ad libitum. However, under a restricted-feeding schedule (food was available from 1400 to 1600 hr; water was always available) lasting for 10 days, a gradual increase of MUA of the LH developed, starting 3-4 hr prior to the feeding time. The elevated MUA lasted up to 6-7 hr after feeding and subsequently returned to the baseline level. This circadian rhythm of MUA of the LH persisted up to 4 days under total food deprivation, with quickly decreasing amplitude after termination of the schedule. MUA rhythm in VMH was less obvious than that in LH. Also, general motor activity showed a rhythm comparable to that of MUA, but it was less prominent. The elevated MUA in the LH prior to the feeding time may have been neural substrate of anticipatory activity appearing under the restricted-feeding schedule. These findings may suggest the existence of a quickly damping oscillator mechanism in the brain, presumably in the LH, which can be induced by daily feeding cues in the absence of the SCN.     

Enhanced effect of daytime restricted feeding on the circadian rhythm of streptozotocin-induced type 2 diabetic rats

There is increasing awareness of the link between impaired circadian clocks and multiple metabolic diseases. However, the impairment of the circadian clock by type 2 diabetes has not been fully elucidated. To understand whether and how the function of circadian clock is impaired under the diabetic condition, we examined not only the expression of circadian genes in the heart and pineal gland but also the behavioral rhythm of type 2 diabetic and control rats in both the nighttime restricted feeding (NRF) and daytime restricted feeding (DRF) conditions. In the NRF condition, the circadian expression of clock genes in the heart and pineal gland was conserved in the diabetic rats, being similar to that in the control rats. DRF shifted the circadian phases of peripheral clock genes more efficiently in the diabetic rats than those in the control rats. Moreover, the activity rhythm of rats in the diabetic group was completely shifted from the dark phase to the light phase after 5 days of DRF treatment, whereas the activity rhythm of rats in the control group was still under the control of the suprachiasmatic nucleus (SCN) after the same DRF treatment. Furthermore, the serum glucose rhythm of type 2 diabetic rats was also shifted and controlled by the external feeding schedule, ignoring the SCN rhythm. Therefore, DRF shows stronger effect on the reentrainment of circadian rhythm in the type 2 diabetic rats, suggesting that the circadian system in diabetes is unstable and more easily shifted by feeding stimuli.     

PINEAL INFLUENCE ON ANNUAL NUCLEAR VOLUME CHANGES IN VENTROMEDIAL HYPOTHALAMIC NUCLEUS (VMH) NEURONS OF THE MALE WISTAR RAT

The ventromedial hypothalamic nucleus (VMH) regulates various autonomic, endocrine, and behavioral activities. These activities show annual changes, and the pineal gland is involved in their adjustment to environmental cues. Therefore, this study investigated whether the VMH belongs to the effector structures of the pineal gland. To abolish the rhythmic melatonin release, male Wistar rats were subjected to pinealectomy (PX) or ganglionectomy (sympathetic denervation of the pineal gland, GX) regularly at the beginning of any of the four seasons. Brains from animals of PX-, GX-, and sham-operated control groups were prepared 3 months later for measurement of the nuclear volume, which changes according to the general gene activity. At each of the four seasons, 2000 nuclei of VMH neurons stemming from 18 animals per group were measured to obtain both seasonal daily mean values and annual mean values,respectively, as well as to calculate annual curves of the nuclear volume using empirical regression and locally adjusted polynomial approximation. The major findings are the following. First,inactivation of the pineal function influences the nuclear activity of VMH neurons. (2) PX and GX mainly depress the nuclear activity, indicating that the pineal influence on the VMH may predominantly be a stimulatory one. Third, size and direction of the changes caused by PX and GX vary in a seasonally dependent manner. Fourth, the annual rhythm of the nuclear activity of the VMH is modified by PX and GX.To explain how the pineal effects on the VMH may be mediated, a possible inhibitory influence of the suprachiasmatic nucleus (SCN),which has been activated in the same animals following both PX and GX, is discussed. In conclusion, the results confirm that the nuclear activity of VMH neurons underlies pineal influences. This also indicates an involvement of the pineal gland in many VMH-regulated functions.(Chronobiology International,17(1),15–28, 2000)     

Circadian rhythm of ERG in Iguana iguana: role of the pineal

In green iguanas, the pineal controls the circadian rhythm of body temperature but not the rhythm of locomotor activity. As part of a program to investigate the characteristics of this multioscillator circadian system, the authors studied the circadian rhythms of the electroretinographic response (ERG) and asked whether the pineal gland is necessary for the expression of this rhythm. ERGs from a total of 24 anesthetized juvenile iguanas were recorded under four different conditions: (a) complete darkness (DD), (b) dim light-dark cycles (dLD), (c) constant dim light (dLL), and (d) pinealectomized in DD. Results demonstrate that the b-wave component of the ERG shows a very clear circadian rhythm in DD and that this rhythm persists in dLL and entrains to dLD cycles. The ERG response is maximally sensitive during the subjective day. Pinealectomy does not abolish the circadian rhythm in ERG, demonstrating that the oscillator responsible for the ERG rhythm is located elsewhere.     

Pineal influence on annual nuclear volume changes in ventromedial hypothalamic nucleus (VMH) neurons of the male Wistar rat

The ventromedial hypothalamic nucleus (VMH) regulates various autonomic, endocrine, and behavioral activities. These activities show annual changes, and the pineal gland is involved in their adjustment to environmental cues. Therefore, this study investigated whether the VMH belongs to the effector structures of the pineal gland. To abolish the rhythmic melatonin release, male Wistar rats were subjected to pinealectomy (PX) or ganglionectomy (sympathetic denervation of the pineal gland, GX) regularly at the beginning of any of the four seasons. Brains from animals of PX-, GX-, and sham-operated control groups were prepared 3 months later for measurement of the nuclear volume, which changes according to the general gene activity. At each of the four seasons, 2000 nuclei of VMH neurons stemming from 18 animals per group were measured to obtain both seasonal daily mean values and annual mean values,respectively, as well as to calculate annual curves of the nuclear volume using empirical regression and locally adjusted polynomial approximation. The major findings are the following. First,inactivation of the pineal function influences the nuclear activity of VMH neurons. (2) PX and GX mainly depress the nuclear activity, indicating that the pineal influence on the VMH may predominantly be a stimulatory one. Third, size and direction of the changes caused by PX and GX vary in a seasonally dependent manner. Fourth, the annual rhythm of the nuclear activity of the VMH is modified by PX and GX.To explain how the pineal effects on the VMH may be mediated, a possible inhibitory influence of the suprachiasmatic nucleus (SCN),which has been activated in the same animals following both PX and GX, is discussed. In conclusion, the results confirm that the nuclear activity of VMH neurons underlies pineal influences. This also indicates an involvement of the pineal gland in many VMH-regulated functions.(Chronobiology International,17(1),15-28, 2000)     

Avian Melatonin Synthesis: Photic and Circadian Regulation of Serotonin N-Acetyltransferase mRNA in the Chicken Pineal Gland and Retina

Abstract: The circadian rhythms in melatonin production in the chicken pineal gland and retina reflect changes in the activity of serotonin N -acetyltransferase (arylalkylamine N -acetyltransferase; AA-NAT; EC 2.3.1.87). Here we determined that the chicken AA-NAT mRNA is detectable in follicular pineal cells and retinal photoreceptors and that it exhibits a circadian rhythm, with peak levels at night. AA-NAT mRNA was not detected in other tissues. The AA-NAT mRNA rhythm in the pineal gland and retina persists in constant darkness (DD) and constant lighting (LL). The amplitude of the pineal mRNA rhythm is not decreased in LL. Light appears to influence the phase of the clock driving the rhythm in pineal AA-NAT mRNA in two ways: The peak is delayed by ∼6 h in LL, and it is advanced by >4 h by a 6-h light pulse late in subjective night in DD. Nocturnal AA-NAT mRNA levels do not change during a 20-min exposure to light, whereas this treatment dramatically decreases AA-NAT activity. These observations suggest that the rhythmic changes in chicken pineal AA-NAT activity reflect, at least in part, clock-generated changes in mRNA levels. In contrast, changes in mRNA content are not involved in the rapid light-induced decrease in AA-NAT activity.     

Food anticipatory activity behavior of mice across a wide range of circadian and non-circadian intervals

When rodents are fed in a limited amount during the daytime, they rapidly redistribute some of their nocturnal activity to the time preceding the delivery of food. In rats, anticipation of a daily meal has been interpreted as a circadian rhythm controlled by a food-entrained oscillator (FEO) with circadian limits to entrainment. Lesion experiments place this FEO outside of the light-entrainable circadian pacemaker in the suprachiasmatic nucleus. Mice also anticipate a fixed daily meal, but circadian limits to entrainment and anticipation of more than 2 daily meals, have not been assessed. We used a video-based behavior recognition system to quantify food anticipatory activity in mice receiving 2, 3, or 6 daily meals at intervals of 12, 8, or 4-hours (h). Individual mice were able to anticipate as many as 4 of 6 daily meals, and anticipation persisted during meal omission tests. On the 6 meal schedule, pre-prandial activity and body temperature were poorly correlated, suggesting independent regulation. Mice showed a limited ability to anticipate an 18 h feeding schedule. Finally, mice showed concurrent circadian and sub-hourly anticipation when provided with 6 small meals, at 30 minute intervals, at a fixed time of day. These results indicate that mice can anticipate feeding opportunities at a fixed time of day across a wide range of intervals not previously associated with anticipatory behavior in studies of rats. The methods described here can be exploited to determine the extent to which timing of different intervals in mice relies on common or distinct neural and molecular mechanisms.     

Circadian rhythms of indoleamines and serotonin N-acetyltransferase activity in the pineal gland

Conclusion The circadian rhythm of melatonin synthesis in the pineal glands of various species has been summarized. The night-time elevation of melatonin content is in most if not all cases regulated by the change of N-acetyltransferase activity. In mammals, the N-acetyltransferase rhythm is controlled by the central nervous system, presumably by suprachiasmatic nuclei in hypothalamus through the superior cervical ganglion. In birds, the circadian oscillator that regulates the N-acetyltransferase rhythm is located in the pineal glands. The avian pineal gland may play a biological clock function to control the circadian rhythms in physiological, endocrinological and biochemical processes via pineal hormone melatonin.     

Physiology of avian circadian pacemakers.

The pineal gland plays a cental role in the circadian organization of birds, although it is clearly only one component in a system with other components that have not yet been positively identified. The relative importance of the pineal and other components may vary from one group of birds to another. In the most thoroughly studied species, the house sparrow, pineal removal abolishes circadian rhythmicity; rhythmicity is restored by transplantation of a donor bird's pineal and the restored rhythm has the phase of the donor. This, and other evidence, argues convincingly that the pineal is a pacemaker in the sparrow circadian system. The pineal of the chicken has circadian rhythms in several biochemical parameters that result in the rhythmic synthesis of melatonin. The activity of one enzyme in this pathway is rhythmic for at least two cycles in organ culture. In view of this result it is interesting that pineal removal does not abolish circadian rhythmicity in chickens. The fact that lesions of the suprachiasmatic nuclei abolish circadian rhythms in sparrows, several mammalian species, and perhaps Japanese quail and reptiles, suggests that vertebrate circadian organization may be based on differentially weighted interactions between the pineal, the suprachiasmatic nuclei, and perhaps other brain regions.     

Effect of BRAND’s Essence of Chicken on the resetting process of circadian clocks in rats subjected to experimental jet lag

BRAND’s Essence of Chicken (BEC) has been widely used as a traditional remedy by people in Southeast Asia, which is proved to have an effect on the central nervous system (CNS) and autonomic nervous system (ANS). However, whether and how BEC consumption may affect mammalian circadian system is still largely unknown. In the present study, we investigated the effect of BEC feeding on the adaptation of circadian clocks to the experimental jet lag in rats. After the 12-h experimental jet lag through extending the light period, BEC feeding markedly facilitated the re-entrainment of all examined clock genes (Bmal1, Cry1, Per1, and Per2) in the pineal gland. The resetting time course of pineal clock genes was reduced from 7 days to only 3–5 days by BEC feeding, which was almost equal to the effect of melatonin feeding. In the liver clock, the facilitating effect of BEC feeding was mainly displayed in the re-entrainment of Bmal1 and Per2 by shortening their resetting processes for nearly 2 days. However, the resetting rate of locomotor activity rhythm was not affected by BEC feeding, suggesting that BEC might be unable to affect the behavioral rhythm.     

Thymidine incorporation by cultured chick pineal glands is not subject to adrenergic regulation

Norepinephrine is known to play a role in regulating the circadian rhythms of serotonin N-acetyltransferase activity and melatonin formation in the chick pineal gland. We have recently demonstrated that the cultured chick pineal exhibits a circadian rhythm in the incorporation of thymidine. In this study we show that this latter rhythm is not subject to adrenergic control.     

Circadian Rhythm in Pineal N-Acetyltransferase Activity: Rapid Phase Reversal and Response to Shorter than 24-Hour Cycles (IV)

N-Acetyltransferase (NAT) is an enzyme whose rhythmic activity in the pineal gland and retina is responsible for circadian rhythms in melatonin. The NAT activity rhythm has circadian properties such as persistence in constant conditions and precise control by light and dark. Experiments are reported in which chicks (Gallus domesticus), raised for 3 weeks in 12 h of light alternating with 12 h of dark (LD12:12), were exposed to 1-3 days of light-dark treatments during which NAT activity was measured in their pineal glands. (a) In LD12:12, NAT activity rose from less than 4.5 nmol/pineal gland/h during the light-time to 25-50 nmol/pineal gland/h in the dark-time. Constant light (LL) attenuated the amplitude of the NAT activity rhythm to 26-45% of the NAT activity cycle in LD12:12 during the first 24 h. (b) The timing of the increase in NAT activity was reset by the first full LD12:12 cycle following a 12-h phase shift of the LD12:12 cycle (a procedure that reversed the times of light and dark by imposition of either 24 h of light or dark). This result satisfies one of the criteria for NAT to be considered part of a circadian driving oscillator. (c) In less than 24-h cycles [2 h of light in alternation with 2 h of dark (LD2:2), 4 h of light in alternation with 4 h of dark (LD4:4), and 6 h of light in alternation with 6 h of dark (LD6:6)], NAT activity rose in the dark during the chicks' previously scheduled dark-time but not the previously scheduled light-time of LD12:12. In a cycle where 8 h of light alternated with 8 h of dark (LD8:8), NAT activity rose in both 8-h dark periods, even though the second one fell in the light-time of the prior LD12:12 schedule.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ontogeny of N-acetyltransferase activity rhythm in pineal gland of chick embryo

1. N-acetyltransferase was present in pineal glands of 14-day-old chick embryos though no rhythm either in LL, DD or LD 12:12 was observed in this age. 2. Daily rhythm in pineal NAT activity was found in 18-day-old embryos incubated under LD 12:12 and LD 16:8 but no NAT rhythm was detected in DD or LL. 3. NAT rhythm persists for 2 days in constant darkness and it may be circadian in nature. 4. Presence of melatonin (85 +/- 8 pg/mg tissue) was detected in pineals of 18-day-old chick embryos.     

Circadian feeding and locomotor rhythms in pigeons and house sparrows.

Feeding and locomotor activities were measured simultaneously in homing pigeons (Columba livia) and house sparrows (Passer domesticus). Feeding, as well as locomotor activity, was found to be regulated by a circadian clock in both of these species. Implantation of melatonin-filled capsules or exposure to constant light abolished feeding and locomotor rhythms in both species. Removal of the pineal gland from pigeons did not abolish either rhythm, whereas pinealectomy abolished both feeding and locomotor rhythms in house sparrows. Although feeding rhythms were generally more robust than locomotor rhythms in both of these species, different feeding and locomotor free-running periods were not observed within any individual pigeon or house sparrow. These results are consistent with the hypothesis that each of these species has a single pacemaker that regulates the timing of feeding and locomotor activity, but they do not rule out the possibility that separate clocks regulate these behaviors.     

60-Hz electric-field effects on pineal melatonin rhythms: time course for onset and recovery

Rats exposed for 3 weeks to uniform 60-Hz electric fields of 39 kV/m (effective field strength) failed to show normal pineal gland circadian rhythms in serotonin N-acetyl transferase activity and melatonin concentrations. The time required for recovery of the melatonin rhythm after cessation of field exposure was determined to be less than 3 days. The rapid recovery suggests that the overall metabolic competence of the pineal is not permanently compromised by electric-field exposure, and that the circadian rhythm effect may be neuronally mediated.     

Prenatal Entrainment of Rat Testicular Malate Dehydrogenase Activity Circadian Rhythm

In mammals the photoperiodic synchronization of circadian system starts before birth. During fetal and neonatal period mothers relay the photoperiodic information to their litter. The maternal pineal melatonin 24 h cycle acts as a synchronizing signal. We have studied the effect of pineal maternal sympathetic denervation and administration of melatonin to mothers denervated during gestation on the prenatal synchronization of testicular malate dehydrogenase (MDH) activity circadian rhythm of the offspring 25 days after birth. When mothers were denervated at the 7th, 10th or 11th day of gestation, pups showed disruption of testicular MDH activity circadian rhythms. In contrast, no disruptive effect was observed when the mothers were denervated on the 12th or 14th day of gestation. When denervated mothers (7th day of gestation) were treated with a daily dose of melatonin from the 11th to the 14th day of gestation, pups showed a MDH activity circadian rhythm. The hormone failed to impose a daily phase when administered from the 9th to the 12th day of gestation. Results suggest that prenatal synchronization in the rat occurs very early in the development, before suprachiasmatic nuclei morphologic arrangement and functional activity begin.     

Relationship of atypical melatonin rhythm with two circadian clock outputs in B6D2F(1) mice

Circadian rhythms in body temperature, locomotor activity, and the circadian changes of plasma and pineal melatonin content were investigated in B6D2F(1) mice synchronized by 12 h of light and 12 h of darkness. During 8 wk continuous recording, activity and temperature displayed a marked stable and reproducible circadian rhythm, with both peaks occurring near the middle of darkness. Both 24- and 12-h rhythmic components were also significantly detected. Mean plasma melatonin concentration rose steadily during the light span and reached a maximum (30.6 +/- 10.0 pg/ml) at 11 h after light onset (HALO), then gradually decreased after the onset of darkness to a nadir (4.7 +/- 0.4 pg/ml) at 20 HALO. Mean pineal content followed a pattern parallel to that of plasma concentration (peak at 11 HALO: 17.7 +/- 1.0 pg/gland; trough at 17 HALO: 4.7 +/- 1.0 pg/gland). In addition, a second sharp peak was observed at 21 HALO (20.2 +/- 3.5 pg/gland). Plasma and pineal contents displayed large and statistically significant circadian changes, with a composite rhythm of period (24 + 12 h). This mouse model has predominant production and secretion of melatonin during the day. This possibly contributes to a similar coupling between chronopharmacology mechanisms and the rest-activity cycle in these mice and in human subjects.     

Circadian rhythms of melatonin in European starlings exposed to different lighting conditions: relationship with locomotor and feeding rhythms

In passerine birds, the periodic secretion of melatonin by the pineal organ represents an important component of the pacemaker that controls overt circadian functions. The daily phase of low melatonin secretion generally coincides with the phase of intense activity, but the precise relationship between the melatonin and the behavioral rhythms has not been studied. Therefore, we investigated in European starlings (Sturnus vulgaris) (1) the temporal relationship between the circadian plasma melatonin rhythm and the rhythms in locomotor activity and feeding; (2) the persistence of the melatonin rhythm in constant conditions; and (3) the effects of light intensity on synchronized and free-running melatonin and behavioral rhythms. There was a marked rhythm in plasma melatonin with high levels at night and/or the inactive phase of the behavioral cycles in almost all birds. Like the behavioral rhythms, the melatonin rhythm persisted for at least 50 days in constant dim light. In the synchronized state, higher daytime light intensity resulted in more tightly synchronized rhythms and a delayed melatonin peak. While all three rhythms usually assumed a rather constant phase relationship to each other, in one bird the two behavioral rhythms dissociated from each other. In this case, the melatonin rhythm retained the appropriate phase relationship with the feeding rhythm. Accepted: 10 December 1999     

Prenatal Entrainment of Rat Testicular Malate Dehydrogenase Activity Circadian Rhythm

In mammals the photoperiodic synchronization of circadian system starts before birth. During fetal and neonatal period mothers relay the photoperiodic information to their litter. The maternal pineal melatonin 24 h cycle acts as a synchronizing signal. We have studied the effect of pineal maternal sympathetic denervation and administration of melatonin to mothers denervated during gestation on the prenatal synchronization of testicular malate dehydrogenase (MDH) activity circadian rhythm of the offspring 25 days after birth. When mothers were denervated at the 7th, 10th or 11th day of gestation, pups showed disruption of testicular MDH activity circadian rhythms. In contrast, no disruptive effect was observed when the mothers were denervated on the 12th or 14th day of gestation. When denervated mothers (7th day of gestation) were treated with a daily dose of melatonin from the 11th to the 14th day of gestation, pups showed a MDH activity circadian rhythm. The hormone failed to impose a daily phase when administered from the 9th to the 12th day of gestation. Results suggest that prenatal synchronization in the rat occurs very early in the development, before suprachiasmatic nuclei morphologic arrangement and functional activity begin.