Maternal Suprachiasmatic Nuclei Research Articles

Challenging the Integrity of Rhythmic Maternal Signals Revealed Gene-Specific Responses in the Fetal Suprachiasmatic Nuclei.

During fetal stage, maternal circadian system sets the phase of the developing clock in the suprachiasmatic nuclei (SCN) via complex pathways. We addressed the issue of how impaired maternal signaling due to a disturbed environmental light/dark (LD) cycle affects the fetal SCN. We exposed pregnant Wistar rats to two different challenges – a 6-h phase shift in the LD cycle on gestational day 14, or exposure to constant light (LL) throughout pregnancy – and detected the impact on gene expression profiles in 19-day-old fetuses. The LD phase shift, which changed the maternal SCN into a transient state, caused robust downregulation of expression profiles of clock genes (Per1, Per2, and Nr1d1), clock-controlled (Dbp) genes, as well as genes involved in sensing various signals, such as c-fos and Nr3c1. Removal of the rhythmic maternal signals via exposure of pregnant rats to LL abolished the rhythms in expression of c-fos and Nr3c1 in the fetal SCN. We identified c-fos as the gene primarily responsible for sensing rhythmic maternal signals because its expression profile tracked the shifted or arrhythmic maternal SCN clock. Pathways related to the maternal rhythmic behavioral state were likely not involved in driving the c-fos expression rhythm. Instead, introduction of a behavioral rhythm to LL-exposed mothers via restricted feeding regime strengthened rhythm in Vip expression in the fetal SCN. Our results revealed for the first time that the fetal SCN is highly sensitive in a gene-specific manner to various changes in maternal signaling due to disturbances of environmental cycles related to the modern lifestyle in humans.

Exposure of Pregnant Rats to Restricted Feeding Schedule Synchronizes the SCN Clocks of Their Fetuses under Constant Light but Not under a Light-Dark Regime

The circadian clock in the suprachiasmatic nucleus (SCN) develops gradually during the prenatal and early postnatal period. In the rat, this period lasts from around the 15th day of gestation until the 10th day of postnatal development. The circadian system of fetuses and newborn pups is entrained mostly by nonphotic maternal cues during prenatal and early postnatal development. The aim of the present study was to ascertain whether exposure of pregnant rats to a restricted feeding (RF) regime was able to entrain the circadian clock in the SCN of their fetuses during the prenatal period. The potency of RF as an entraining cue was tested under conditions when pregnant rats were entrained to an external light/dark (LD) cycle as well as under conditions when the external timing signal was lacking, i.e., under constant light (LL). The control groups were fed ad libitum and the experimental groups had restricted access to food for 6 h during their resting time throughout pregnancy. Daily profiles of Avp and c-fos gene expression were examined by in situ hybridization in the SCN of 1-day-old pups. The data demonstrated that RF in pregnant rats kept under LD cycle did not notably affect the daily rhythms of c-fos and Avp expression in the SCN of pups. The SCN profiles of Avp and c-fos gene expression did not exhibit circadian rhythms in pups born to mothers maintained in LL and fed ad libitum, likely due to desynchrony among the pups within a litter. However, RF in the pregnant rats kept under LL restored the circadian rhythmicity of c-fos and Avp expression in the SCN of their newborn pups. The results suggest that the fetal SCN clock is dominantly entrained by rhythmic signals from the maternal SCN. However, under conditions when the rhythmic signaling might be lacking, such as LL, regular food intake of the mothers may also play an important role in synchronization of the fetal SCN clock during prenatal ontogenesis.

Maternal Control of the Fetal and Neonatal Rat Suprachiasmatic Nucleus

The molecular clockwork underlying the generation of circadian rhythmicity within the suprachiasmatic nucleus (SCN) develops gradually during ontogenesis. The authors' previous work has shown that rhythms in clock gene expression in the rat SCN are not detectable at embryonic day (E) 19, start to form at E20 and develop further via increasing amplitude until postnatal day (P) 10. The aim of the present work was to elucidate whether and how swiftly the immature fetal and neonatal molecular SCN clocks can be reset by maternal cues. Pregnant rats maintained under a light-dark (LD) regimen with 12 h of light and 12 h of darkness were exposed to a 6-h delay of the dark period and released into constant darkness at different stages of the fetal SCN development. Adult rats maintained under the same LD regimen were exposed to an identical shifting procedure. Daily rhythms in spontaneous c-fos, Avp, Per1, and Per2 expression were examined within the adult and newborn SCN by in situ hybridization. Exposure of adult rats to the shifting procedure induced a significant phase delay of locomotor activity within 3 days after the phase shift as well as a delay in the rhythms of c-fos and Avp expression within 3 days and Per1 and Per2 expression within 5 days. Exposure of pregnant rats to the shifting procedure at E18, but not at E20, delayed the rhythm in c-fos and Avp expression in the SCN of newborn pups at P0-1. The shifting procedure at E20 did, however, induce a phase delay of Per1 and Per2 expression rhythms at P3 and P6. Hence, 5 days were necessary for phase-shifting the pups' SCN clock by maternal cues, be it the interval between E18 and P0-1 or the interval between E20 and P3, while only 3 days were necessary for phase-shifting the maternal SCN by photic cues. These results demonstrate that the SCN clock is capable of significant phase shifts at fetal developmental stages when no or very faint molecular oscillations can be detected.

Maternal Feeding Controls Fetal Biological Clock

BackgroundIt is widely accepted that circadian physiological rhythms of the fetus are affected by oscillators in the maternal brain that are coupled to the environmental light-dark (LD) cycle.Methodology/Principal FindingsTo study the link between fetal and maternal biological clocks, we investigated the effects of cycles of maternal food availability on the rhythms of Per1 gene expression in the fetal suprachiasmatic nucleus (SCN) and liver using a transgenic rat model whose tissues express luciferase in vitro. Although the maternal SCN remained phase-locked to the LD cycle, maternal restricted feeding phase-advanced the fetal SCN and liver by 5 and 7 hours respectively within the 22-day pregnancy.Conclusions/SignificanceOur results demonstrate that maternal feeding entrains the fetal SCN and liver independently of both the maternal SCN and the LD cycle. This indicates that maternal-feeding signals can be more influential for the fetal SCN and particular organ oscillators than hormonal signals controlled by the maternal SCN, suggesting the importance of a regular maternal feeding schedule for appropriate fetal molecular clockwork during pregnancy.

Entrainment of Syrian hamster circadian activity rhythms by neonatal melatonin injections.

The circadian rhythms of fetal and neonatal rodents are entrained by their mother. This entrainment is dependent upon the maternal suprachiasmatic nucleus (SCN), but the mechanism of entrainment is unknown. Administration of the pineal hormone melatonin to pregnant, SCN-lesioned female Syrian hamsters entrains the activity rhythms of their hamster pups. The aim of this study was to determine whether melatonin injected directly in neonatal Syrian hamsters is able to entrain circadian rhythms and, if so, for how long this effect persists during development. Injection of melatonin in two groups of hamster pups at opposite phases on postnatal days 1-5 entrained the onset of activity rhythms on the day of weaning to two phases 10.67 h apart. Melatonin injection did not entrain activity rhythms to opposite phases on either postnatal days 6-10 or 21-25. Vehicle injection did not entrain animals to opposite phases at any of the ages studied. These results demonstrate that melatonin is able to act directly on the neonate to cause entrainment and that this effect disappears after postnatal day 6.

C-fos Expression in Fetal and Maternal Suprachiasmatic Nucleus (SCN) in the Sheep

Circadian rhythms (CR) are changes in physiological variables with a period close to 24 h., synchronized by environment signals. The fetal sheep presents CR of plasma prolactin and cerebospinal fluid vasopressin at 135 days of gestational age (GA, term 147 days) In adults, CR are driven by the SCN. We investigated whether the maternal and fetal sheep SCN behave as circadian oscillators. To that effect, we measured expression of c-fos (marker of cellular activity) at 135 days's GA in the SCNs of 6 maternal sheep and their fetuses (n=9) at 1200h (day) and 2400h (night). The sheep. were sacrificed with pentobarbitone and the fetuses were delivered by hysterotomy. The maternal and fetal brain were perfused with 4% paraformaldehyde in phosphate buffer 0.1 M, pH 7.4. The brains were removed, post-fixed and the SCN cut in 40μm frozen sections. Alternated sections were stained with cresyl violet of with anti-Fos antibody (Oncogene Science), developed with avidin-biotin and diaminobenzidine. We measured the density of Fos immunoreactive (Fos +) neurons bys mm2 in the SCN. Result were mean ± SE; ANOVA.

D1-dopamine receptors activate c-fos expression in the fetal suprachiasmatic nuclei.

The existence of an activatable dopamine system within the hypothalamic suprachiasmatic nuclei (SCN), the site of a biological clock, was investigated in rats during fetal life. In situ hybridization studies revealed that D1-dopamine receptor mRNA was highly expressed in the fetal SCN and not expressed in other hypothalamic regions. Cocaine injected into pregnant rats or directly into rat fetuses on day 20 of gestation selectively activated c-fos gene expression in the fetal SCN; cocaine did not induce c-fos expression elsewhere in the fetal brain or in the maternal SCN. This cocaine-induced activation of c-fos expression in fetal SCN was mediated in part through D1-dopamine receptors, as the cocaine-induced activation was partially blocked by the D1-dopamine receptor antagonist SCH 23390. In addition, the selective D1-dopamine receptor agonist SKF 38393 induced high levels of c-fos expression in the fetal SCN. The presence of an activatable dopamine system within the fetal SCN provides a mechanism through which maternal signals could entrain the fetal biological clock and through which maternally administered psychotropic drugs could alter normal development of the circadian timing system.

Periodic feeding of SCN-lesioned pregnant rats entrains the fetal biological clock

Destruction of the maternal suprachiasmatic nuclei (SCN) early in gestation disrupts maternal communication of time-of-day information to the rat fetus. In the present study, we demonstrated that periodic feeding (food cue) to SCN-lesioned pregnant rats entrains the fetal biological clock. The phase of the drinking behavior rhythm was examined in pups reared in constant darkness, beginning at weaning. In several control (uncued) litters, pup phases at weaning were scattered. In other control litters where within-litter coordination of phase was observed, the average litter phase was unpredictable. In contrast, drinking rhythms of pups whose dams had received food cue during gestation were synchronized within- and between-litters, suggesting that prenatal food cue entrained the fetuses. The effect of food cue occurs prenatally, as similar results were obtained when offspring of SCN-lesioned, cued dams were fostered to lesioned, uncued dams on the day of birth. The present results, along with data from this and other laboratories, suggest that redundant mechanisms communicate time-of-day information to the fetus.

Development of hamster circadian rhythms: role of the maternal suprachiasmatic nucleus.

During development, the circadian rhythms of rodents become entrained to rhythmicity of the mother. Rhythms in behavior and in neuroendocrine function are regulated by a circadian pacemaker thought to be located within the suprachiasmatic nucleus (SCN) of the hypothalamus. Evidence indicates that this pacemaker begins to function and to be entrained by maternal rhythms before birth. Although the maternal rhythms which mediate prenatal entrainment of the fetal circadian pacemaker have not been identified, it is likely that they are regulated by the maternal SCN. The role of the maternal SCN in entrainment of the offspring was examined in Syrian hamsters (Mesocricetus auratus) by measuring the activity/rest rhythms of pups. Using the synchrony among the rhythms of pups within a litter as an indication that the pups had been entrained, the effect on entrainment of ablating the maternal SCN was determined. Lesions of the maternal SCN which were performed early in gestation (day 7) and which destroyed at least 75% of the SCN were found to disrupt the normal within litter synchrony among pups, indicating interference with the normal mechanism of entrainment. The effect of lesions on day 7 of gestation could mean that the maternal SCN is important for entrainment of the pups before birth, after birth, or during both of these times. To determine if the maternal SCN is specifically important for prenatal entrainment, lesions were performed two days before birth on day 14 of gestation. Lesions of the maternal SCN on day 14 were not as disruptive as were lesions on day 7. This suggests that the maternal SCN is important between days 7 and 14 of gestation and that the synchrony normally observed at weaning is already established, in part, on or before day 14 of gestation. This further suggests that an entrainable circadian pacemaker is present in the fetus only two weeks after fertilization.

Entrainment of hamster pup circadian rhythms by prenatal melatonin injections to the mother.

A circadian pacemaker, thought to be within the suprachiasmatic nucleus (SCN) of the hypothalamus, begins to function before birth in rodents. Prenatal entrainment of the pacemaker appears to be mediated by signals regulated by the maternal SCN; ablation of the mother's SCN during gestation disrupts the normal phase of the pups' rhythms. The present paper presents an experimental approach for identifying candidate entraining signals and for testing when they are effective during development. The candidate signal examined in these experiments was the pineal gland hormone, melatonin. Female golden hamsters (Mesocricetus auratus) received SCN lesions on day 7 of gestation. During the last week of gestation, they were given two daily subcutaneous injections of oil 12 h apart. One of the injections each day contained melatonin (10, 50, or 100 micrograms). The phases of the pups' activity rhythms were measured at weaning and were found to be related to the timing of the daily injection that contained melatonin, demonstrating that the melatonin directly or indirectly set the phase of the pups' rhythms. Injections given over 4 days of gestation were found to be as effective as injections given over 7 days. Although a physiological role for melatonin as an entraining signal has not been demonstrated, the results show that exogenous, prenatal treatment can predictably set the phase of the offsprings' circadian rhythms.

Development of a fetal circadian rhythm after disruption of the maternal circadian system

The role of maternal circadian rhythms in the development of the fetal circadian system was investigated in the rat. Pregnant females were subjected to procedures known to disrupt circadian function, ablation of the maternal suprachiasmatic nuclei (SCN) or housing in constant illumination, on gestational day 10. Circadian function was assessed in fetuses at gestational day 22 by analysis of glucose utilization in hypothalamic slices in vitro using the 2-deoxyglucose method. Fetuses from control females exhibit a robust rhythm in glucose utilization in the SCN. In contrast, the SCN of fetuses from females with SCN lesions, or housed in constant illumination, show no significant day-night difference in glucose utilization. Analysis of individual brains indicates, however, that this apparent disruption in the development of circadian rhythmicity in metabolism in the fetal SCN is due to a desynchronization of individual fetuses resulting from the loss of maternal entraining influences. Thus, the fetal SCN is capable of developing a circadian rhythm in glucose utilization independent of the maternal circadian system.

THE CIRCADIAN-GATED TIMING OF BIRTH IN RATS: DISRUPTION BY MATERNAL SCN LESIONS OR BY REMOVAL OF THE FETAL BRAIN

In rats, the hour of birth is gated over a 36-hr temporal window by the phase of the daily light-dark cycle during pregnancy. We have previously shown that the suprachiasmatic nuclei (SCN), the site of a known circadian pacemaker, are oscillating in phase with the prevailing light-dark cycle in the fetus. Since the onset of parturition is governed by the fetal brain in some species, we have speculated that a possible role of a functioning and entrainable circadian clock during fetal life is that it might be involved in the circadian-gated initiation of parturition. First, we showed the circadian gating of birth in our animals by exposing different groups of dams to lighting cycles of opposite phase during pregnancy. Regardless of the phase of the prenatal lighting cycle, the time of birth was gated over a 36-hr temporal window so that most births occurred during the daytime hours. Next, we found that destruction of the maternal SCN (on day 7 of gestation) eliminated the circadian gating; births occurred in a single distribution that peaked in the middle of the 36-hr window. Finally, removal of all the fetal brains from each litter also disrupted the circadian gating of birth; dams of brain-aspirated fetuses no longer exhibited a daytime preference for births. These results show that the maternal SCN are necessary for the normal circadian gating of birth and are also consistent with a role for the fetal brain (and possibly the fetal SCN) in this process. Support by HD14427.

The circadian-gated timing of birth in rats: disruption by maternal SCN lesions or by removal of the fetal brain

The roles of the maternal suprachiasmatic nuclei (SCN) and fetal brain in the circadian-gated timing of birth were studied in rats. The circadian gating of birth was shown by exposing different groups of dams to lighting cycles of opposite phase during pregnancy. Regardless of the phase of the prenatal lighting cycle, the time of birth was gated over a 36-h temporal window so that most births occurred during subjective day. Destruction of the maternal SCN eliminated the circadian gating; births occurred in a single distribution that peaked in the middle of the 36-h window. Removal of the fetal brain also disrupted the circadian gating of birth; dams of brain-aspirated fetuses no longer exhibited a daytime preference for births. These results show that the maternal SCN are necessary for the normal circadian gating of birth and are also consistent with a role for the fetal brain in this process.

Maternal suprachiasmatic nuclei are necessary for maternal coordination of the developing circadian system

During late fetal and early neonatal life, the maternal circadian system coordinates the timing of a circadian clock in the hypothalamic suprachiasmatic nuclei (SCN) to the prevailing light-dark cycle. The role of the maternal SCN in the process of maternal coordination was investigated in rats. Complete lesions of the maternal SCN on day 7 of gestation disrupted rhythms of SCN glucose utilization in fetuses and pineal N-acetyltransferase activity in 10-d-old pups. This disruption was probably due to the desynchronization of individually oscillating fetal SCN, because individual pups born to and reared by SCN-lesioned dams under constant conditions exhibited normal circadian rhythms in drinking behavior. Cross-foster studies showed that the maternal circadian system can coordinate developing circadian rhythmicity during either the pre- or postnatal period. The results indicate that the maternal SCN are a necessary component of the mechanism of maternal coordination during both the pre- and postnatal periods.

Photic influences on the developing mammal.

In adult mammals, the daily light-dark cycle acts via the retinohypothalamic pathway to entrain the circadian clock in the suprachiasmatic nuclei (SCN) and to communicate information about daylength to photoperiodic species. Studies in rats show that during late fetal and early neonatal life, before the retinohypothalamic pathway has innervated the SCN, the maternal circadian system entrains the timing of the developing clock to prevailing lighting conditions. Although the nature of the maternal output signal(s) used to entrain the developing clock has not been elucidated, the maternal SCN are a necessary component of maternal entrainment during both prenatal and postnatal life. Maternal entrainment of the fetal and neonatal clock thus ensures that the developing circadian system is synchronized to the outside world until maturation of the retinohypothalamic pathway permits direct photic entrainment. The maternal circadian system is not only necessary for entrainment of the developing circadian system, but recent studies suggest it may also provide the immature mammal with important photoperiodic information. In the montane vole (Microtus montanus) and the Djungarian hamster (Phodopus sungorus), the prenatal photoperiod affects postnatal photoperiodic responses, and cross-fostering experiments show that this information about daylength is perceived by the fetus. This prenatal information, in conjunction with postnatal perception of photoperiod, allows the developing animal to determine which way the season is changing and to modify the rate of reproductive maturation accordingly.

ABLATION OF THE MATERNAL SUPRACHIASMATIC NUCLEI DISRUPTS THE TIMING OF THE FETAL CIRCADIAN CLOCK

We have proven in the rat that a functional circadian clock oscillates in the fetal suprachiasmatic nuclei (SCN) and that the maternal circadian system coordinates the phase of the fetal clocks to ambient lighting conditions (Science 220:969, 1983). As the first step towards elucidating the mechanism of maternal coordination, we examined the effects of maternal SCN ablation. The 14C-deoxyglucose (DG) method was used to monitor metabolic activity of the fetal SCN. Timed pregnant rats were exposed to diurnal lighting throughout pregnancy. On gestational day 7, dams either received bilateral electrolytic SCN lesions or sham lesions. On gestational day 18, all were placed in constant darkness; dams were injected i.v. with 145 μCi/kg DG during either mid-subjective day or mid-subjective night on gestational day 21. Autoradiographs of serial sections of fetal brains from sham-operated dams revealed that the fetal SCN were all metabolically active during subjective day (n=18) and all inactive during subjective night (n=18). In contrast, after histologically confirmed complete SCN ablation in the dam, there was no significant day-night variation of SCN metabolic activity in the fetuses; activity was scattered over the inactive to active range within each of the 3 litters studied (8-10 fetuses/litter) at each of the two injection times. The results indicate that the maternal SCN are essential for coordination of the fetal circadian clock. (Supported by PHS Grant HD14427)

Effects of elimination of maternal circadian rhythms during pregnancy on the postnatal development of circadian corticosterone rhythm in blinded infantile rats.

The influence of maternal circadian rhythms on fetal circadian oscillations during pregnancy was examined. Circadian rhythms of spontaneous locomotor activity and plasma corticosterone level in pregnant rats were eliminated by bilateral lesions of the suprachiasmatic nuclei (SCN) at different stages of gestation and the postnatal manifestation of the circadian corticosterone rhythm was examined in individual pups. Effective lesions of SCN at day 3 of gestation resulted in an abortion in all rats examined. Rats whose SCN were lesioned at day 10 or 17 of gestation maintained their pregnancies. At term, pups were removed by Cesarean operation and immediately blinded by bilateral ocular enucleation; they were reared by unoperated foster mother afterwards. All pups from SCN lesioned mothers showed a clear free running circadian rhythm of plasma corticosterone levels after the 4th week of postnatal life. Furthermore, the circadian rhythm that developed in pups from SCN-lesioned mother at day 10 of gestation (G10 pups) was always phase-delayed about 4 h as compared with that in pups from mothers whose SCN were lesioned at day 17 of gestation (G17 pups) and in pups from sham-operated mothers (S pups). It is concluded that the circadian hormone rhythmicity develops in normal fashion postnatally when the maternal SCN are effectively lesioned after day 10 of gestation. The phase-angle difference in the circadian rhythm between G10 pups and G17 or S pups suggests that fetal circadian oscillation is entrainable at least after day 10 of gestation.