Early development of circadian rhythmicity in the suprachiamatic nuclei and pineal gland of teleost, flounder (Paralichthys olivaeus), embryos.

Circadian rhythms enable organisms to coordinate multiple physiological processes and behaviors with the earth's rotation. In mammals, the suprachiasmatic nuclei (SCN), the sole master circadian pacemaker, has entrainment mechanisms that set the circadian rhythm to a 24-h cycle with photic signals from retina. In contrast, the zebrafish SCN is not a circadian pacemaker, instead the pineal gland (PG) houses the major circadian oscillator. The SCN of flounder larvae, unlike that of zebrafish, however, expresses per2 with a rhythmicity of daytime/ON and nighttime/OFF. Here, we examined whether the rhythm of per2 expression in the flounder SCN represents the molecular clock. We also examined early development of the circadian rhythmicity in the SCN and PG. Our three major findings were as follows. First, rhythmic per2 expression in the SCN was maintained under 24 h dark (DD) conditions, indicating that a molecular clock exists in the flounder SCN. Second, onset of circadian rhythmicity in the SCN preceded that in the PG. Third, both 24 h light (LL) and DD conditions deeply affected the development of circadian rhythmicity in the SCN and PG. This is the first report dealing with the early development of circadian rhythmicity in the SCN in fish.

Development and entrainment of the colonic circadian clock during ontogenesis.

Colonic morphology and function change significantly during ontogenesis. In mammals, many colonic physiological functions are temporally controlled by the circadian clock in the colon, which is entrained by the central circadian clock in the suprachiasmatic nuclei (SCN). The aim of this present study was to ascertain when and how the circadian clock in the colon develops during the perinatal period and whether maternal cues and/or the developing pup SCN may influence the ontogenesis of the colonic clock. Daily profiles of clock genes Per1, Per2, Cry1, Cry2, Rev-erbα, Bmal1, and Clock expression in the colon underwent significant modifications since embryonic day 20 (E20) through postnatal days (P) 2, 10, 20, and 30 via changes in the mutual phasing among the individual clock gene expression rhythms, their relative phasing to the light-dark regime, and their amplitudes. An adult-like state was achieved around P20. The foster study revealed that during the prenatal period, the maternal circadian phase may partially modulate development of the colonic clock. Postnatally, the absence and/or presence of rhythmic maternal care affected the phasing of the clock gene expression profiles in pups at P10 and P20. A reversal in the colonic clock phase between P10 and P20 occurred in the absence of rhythmic signals from the pup SCN. The data demonstrate ontogenetic maturation of the colonic clock and stress the importance of prenatal and postnatal maternal rhythmic signals for its development. These data may contribute to the understanding of colonic function-related diseases in newborn children.

Development, maturation, and necessity of transcription factors in the mouse suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) of the hypothalamus is the master mammalian circadian clock. The SCN is highly specialized because it is responsible for generating a near 24 h rhythm, integrating external cues, and translating the rhythm throughout the body. Currently, our understanding of the developmental origin and genetic program involved in the proper specification and maturation of the SCN is limited. Herein, we provide a detailed analysis of transcription factor (TF) and developmental-gene expression in the SCN from neurogenesis to adulthood in mice (Mus musculus). TF expression within the postmitotic SCN was not static but rather showed specific temporal and spatial changes during prenatal and postnatal development. In addition, we found both global and regional patterns of TF expression extending into the adult. We found that the SCN is derived from a distinct region of the neuroepithelium expressing a combination of developmental genes: Six3, Six6, Fzd5, and transient Rx, allowing us to pinpoint the origin of this region within the broader developing telencephalon/diencephalon. We tested the necessity of two TFs in SCN development, RORα and Six3, which were expressed during SCN development, persisted into adulthood, and showed diurnal rhythmicity. Loss of RORα function had no effect on SCN peptide expression or localization. In marked contrast, the conditional deletion of Six3 from early neural progenitors completely eliminated the formation of the SCN. Our results provide the first description of the involvement of TFs in the specification and maturation of a neural population necessary for circadian behavior.

Circadian rhythms in the pineal organ persist in zebrafish larvae that lack ventral brain.

BACKGROUND: The mammalian suprachiasmatic nucleus (SCN), located in the ventral hypothalamus, is a major regulator of circadian rhythms in mammals and birds. However, the role of the SCN in lower vertebrates remains poorly understood. Zebrafish cyclops (cyc) mutants lack ventral brain, including the region that gives rise to the SCN. We have used cyc embryos to define the function of the zebrafish SCN in regulating circadian rhythms in the developing pineal organ. The pineal organ is the major source of the circadian hormone melatonin, which regulates rhythms such as daily rest/activity cycles. Mammalian pineal rhythms are controlled almost exclusively by the SCN. In zebrafish and many other lower vertebrates, the pineal has an endogenous clock that is responsible in part for cyclic melatonin biosynthesis and gene expression. RESULTS: We find that pineal rhythms are present in cyc mutants despite the absence of an SCN. The arginine vasopressin-like protein (Avpl, formerly called Vasotocin) is a peptide hormone expressed in and around the SCN. We find avpl mRNA is absent in cyc mutants, supporting previous work suggesting the SCN is missing. In contrast, expression of the putative circadian clock genes, cryptochrome 1b (cry1b) and cryptochrome 3 (cry3), in the brain of the developing fish is unaltered. Expression of two pineal rhythmic genes, exo-rhodopsin (exorh) and serotonin-N-acetyltransferase (aanat2), involved in photoreception and melatonin synthesis, respectively, is also similar between cyc embryos and their wildtype (WT) siblings. The timing of the peaks and troughs of expression are the same, although the amplitude of expression is slightly decreased in the mutants. Cyclic gene expression persists for two days in cyc embryos transferred to constant light or constant dark, suggesting a circadian clock is driving the rhythms. However, the amplitude of rhythms in cyc mutants kept in constant conditions decreased more quickly than in their WT siblings. CONCLUSION: Our data suggests that circadian rhythms can be initiated and maintained in the absence of SCN and other tissues in the ventral brain. However, the SCN may have a role in regulating the amplitude of rhythms when environmental cues are absent. This provides some of the first evidence that the SCN of teleosts is not essential for establishing circadian rhythms during development. Several SCN-independent circadian rhythms have also been found in mammalian species. Thus, zebrafish may serve as a model system for understanding how vertebrate embryos coordinate rhythms that are controlled by different circadian clocks.

Alterations induced by chronic lead exposure on the cells of circadian pacemaker of developing rats.

Lead (Pb) exposure alters the temporal organization of several physiological and behavioural processes in which the suprachiasmatic nucleus (SCN) of the hypothalamus plays a fundamental role. In this study, we evaluated the effects of chronic early Pb exposure (CePbe) on the morphology, cellular density and relative optical density (OD) in the cells of the SCN of male rats. Female Wistar rats were exposed during gestation and lactation to a Pb solution containing 320 ppm of Pb acetate through drinking water. After weaning, the pups were maintained with the same drinking water until sacrificed at 90 days of age. Pb levels in the blood, hypothalamus, hippocampus and prefrontal cortex were significantly increased in the experimental group. Chronic early Pb exposure induced a significant increase in the minor and major axes and somatic area of vasoactive intestinal polypeptide (VIP)- and vasopressin (VP)-immunoreactive neurons. The density of VIP-, VP- and glial fibrillary acidic protein (GFAP)-immunoreactive cells showed a significant decrease in the experimental group. OD analysis showed a significant increase in VIP neurons of the experimental group. The results showed that CePbe induced alterations in the cells of the SCN, as evidenced by modifications in soma morphology, cellular density and OD in circadian pacemaker cells. These findings provide a morphological and cellular basis for deficits in circadian rhythms documented in Pb-exposed animals.

Differential maturation of circadian rhythms in clock gene proteins in the suprachiasmatic nucleus and the pars tuberalis during mouse ontogeny.

Circadian rhythms of many body functions in mammals are controlled by a master pacemaker, residing in the hypothalamic suprachiasmatic nucleus (SCN), which synchronises peripheral oscillators. The SCN and peripheral oscillators share several components of the molecular clockwork and comprise transcriptional activators (BMAL1 and CLOCK/NPAS2) and inhibitors (mPER1/2 and mCRY1/2). Here we compared the ontogenetic maturation of the clockwork in the SCN and pars tuberalis (PT). The PT is a peripheral oscillator that strongly depends on rhythmic melatonin signals. Immunoreactions for clock gene proteins were determined in the SCN and PT at four different timepoints during four differential stages of mouse ontogeny: foetal (embryonic day 18), newborn (2-day-old), infantile (10-day-old), and adult. In the foetal SCN, levels of immunoreactions of all clock proteins were significantly lower than adult levels except for BMAL1. In the newborn SCN the clock protein immunoreactions had not yet reached adult levels, but the infantile SCN showed similar levels of immunoreactions as the adult. In contrast, immunoreactions for all clock gene proteins in the foetal PT were as intense as in newborn, infantile and adult, and showed the same phase. As the foetal pineal gland is not yet capable of rhythmic melatonin production, the rhythms in clock gene proteins in the foetal PT are presumably dependent on the maternal melatonin signal. Thus, our data provide the first evidence that maternal melatonin is important for establishing and maintaining circadian rhythms in a foetal peripheral oscillator.

Transplanted suprachiasmatic nucleus determines circadian period.

The pacemaker role of the suprachiasmatic nucleus in a mammalian circadian system was tested by neural transplantation by using a mutant strain of hamster that shows a short circadian period. Small neural grafts from the suprachiasmatic region restored circadian rhythms to arrhythmic animals whose own nucleus had been ablated. The restored rhythms always exhibited the period of the donor genotype regardless of the direction of the transplant or genotype of the host. The basic period of the overt circadian rhythm therefore is determined by cells of the suprachiasmatic region.

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.

Development of the mouse suprachiasmatic nucleus: determination of time of cell origin and spatial arrangements within the nucleus.

The suprachiasmatic nucleus (SCN) in mammals functions as the principal circadian pacemaker synchronizing diverse physiological and behavioral processes to environmental stimuli. It consists of heterogeneous populations of cells with unique spatial organization that can vary among species, but are commonly discussed within a framework of two principal regions, the ventrolateral or dorsomedial halves of the nucleus or in other instances the core and shell. In both hamsters and rats, cells of different SCN regions have been shown to have different developmental histories. Using bromodeoxyuridine as a marker of cell division, the present study investigated the time of SCN cell origin in mice (C57BL/6) and their settling patterns within the nucleus. Results show that SCN cytogenesis occurs between embryonic days 12 and 15 and is complete 5 days prior to birth. Cells born on embryonic day 12 are mainly confined to a ventrolateral region of the mid-SCN, whereas cells produced later on embryonic days 13.5 and 14.5 form a cap around the cells produced first and extend into the posterior and anterior ends of the nucleus. These results suggest an ordered spatiotemporal program of SCN cytogenesis whereby a mid-SCN core is born first followed by a surrounding shell of later-born cells. Variations in cytogenesis could affect the relative sizes of different SCN regions and, thereby, affect its function. The relative contributions of a highly ordered program of cytogenesis and intercellular interactions after postmitotic cells leave the germinal epithelium remain to be determined.

Circadian molecular clocks tick along ontogenesis.

The circadian system controls the timing of behavioral and physiological functions in most organisms studied. The review addresses the question of when and how the molecular clockwork underlying circadian oscillations within the central circadian clock in the suprachiasmatic nuclei of the hypothalamus (SCN) and the peripheral circadian clocks develops during ontogenesis. The current model of the molecular clockwork is summarized. The central SCN clock is viewed as a complex structure composed of a web of mutually synchronized individual oscillators. The importance of development of both the intracellular molecular clockwork as well as intercellular coupling for development of the formal properties of the circadian SCN clock is also highlighted. Recently, data has accumulated to demonstrate that synchronized molecular oscillations in the central and peripheral clocks develop gradually during ontogenesis and development extends into postnatal period. Synchronized molecular oscillations develop earlier in the SCN than in the peripheral clocks. A hypothesis is suggested that the immature clocks might be first driven by external entraining cues, and therefore, serve as "slave" oscillators. During ontogenesis, the clocks may gradually develop a complete set of molecular interlocked oscillations, i.e., the molecular clockwork, and become self-sustained clocks.

[Effects of ethanol on the development of circadian time keeping system].

Ethanol exposure during gestation can have devastating consequences on the developing organism. Children who have a history of prenatally exposure to ethanol may show morphological and functional alterations, referred to as fetal alcohol spectrum disorders (FASD). Fetal alcohol syndrome (FAS), which is characterized by pre- and postnatal growth deficiency, specific cranial/facial features, and dysfunction of central nervous system, is the most severe end of FASD. FAS or FASD children are known to suffer from disturbance of sleep and/or food intake behaviors. These neuropsychiatric symptoms may be due to impairment of the system regulating circadian rhythms. Recently, animal studies revealed that ethanol exposure during brain development can cause alterations in the circadian rhythm and its regulating system. We examined the effects of pre- or postnatal exposure to ethanol on the circadian rhythm in adulthood by measuring deep body temperature and wheel running activity in rats. After a phase delay in the light/dark cycle, ethanol-exposed rats took longer than control rats to resynchronize to the new light/dark cycle. These results suggest that both pre- and postnatal ethanol exposure impair the development of the circadian clock response to light cue. Because abnormal development of the circadian clock system might contribute to the neuropsychiatric symptoms seen in FASD, it is believed that normalizing the disturbed rhythm improves the symptoms. However, the mechanisms of dysfunction and potential interventions for disturbance of circadian clock system still remain to be elucidated. Further investigations are required to fully understand long-term effects of ethanol on the development of circadian rhythms.

Expression of clock and clock-driven genes in the rat suprachiasmatic nucleus during late fetal and early postnatal development.

The SCN as a site of the circadian clock itself exhibits rhythmicity. A molecular clockwork responsible for the rhythmicity consists of clock genes and their negative and positive transcriptional-translational feedback loops. The authors' previous work showed that rhythms in clock gene expression in the rat SCN were not yet detectable at embryonic day (E) 19 but were already present at postnatal day (P) 3. The aim of the present study was to elucidate when during the interval E19-P3 the rhythms start to develop in clock gene expression and in clock-controlled, namely in arginine-vasopressin (AVP), gene expression. Daily profiles of Per1, Per2, Cry1, Bmal1, and Clock mRNA and of AVP heteronuclear (hn) RNA as an indicator of AVP gene transcription were assessed in the SCN of fetuses at E20 and of newborn rats at P1 and P2 by the in situ hybridization method. At E20, formation of a rhythm in Per1 expression was indicated, but no rhythms in expression of other clock genes or of the AVP gene were detected. At P1, rhythms in Per1, Bmal1, and AVP and a forming rhythm in Per2 but no rhythm in Cry1 expression were present in the SCN. The Per1 mRNA rhythm was, however, only slightly pronounced. The Bmal1 mRNA rhythm, although pronounced, exhibited still an atypical shape. Only the AVP hnRNA rhythm resembled that of adult rats. At P2, marked rhythms of Per1, Per2, and Bmal1 and a forming rhythm of Cry1, but not of Clock, expression were present. The data suggest that rhythms in clock gene expression for the most part develop postnatally and that other mechanisms besides the core clockwork might be involved in the generation of the rhythmic AVP gene expression in the rat SCN during early ontogenesis.

The rat circadian clockwork and its photoperiodic entrainment during development.

The mammalian circadian pacemaker is located in the suprachiasmatic nucleus (SCN), which is composed of dorsomedial (dm) and ventrolateral (vl) regions. The molecular clockwork responsible for the SCN rhythmicity consists of clock genes and their transcriptional-translational feedback loops. The rat SCN rhythmicity and clockwork are affected by the photoperiod. The aim of this study was to elucidate development of the rat SCN rhythmicity, namely of the rhythmicity of the dm- and vl-SCN and of expression of clock genes and to ascertain when the photoperiod starts to affect the SCN rhythmicity. Rhythmicity of the dm-SCN, measured as the rhythm in spontaneous c-FOS production, developed earlier than that of the vl-SCN, which was measured as the rhythm in c-FOS photoinduction. However, photoperiodic affection of the rhythmicity occurred earlier in the vl-SCN than in the dm-SCN. From the 4 clock genes (Per1, Per2, Cry1 and Bmal1) studied, the expression of Bmal1 and Per1 was rhythmic already in 1-day-old rats; at this age, the Per2 mRNA rhythm just started to form and no rhythm in Cry1 expression was detected. After the second postnatal day, all 4 genes were expressed in a rhythmic manner. Thereafter, the rhythms matured gradually via increasing amplitude. Per1 and Per2 mRNA rhythms started to be affected by the photoperiod at the 10th postnatal day. The data suggest that the rhythms in clock genes expression in the rat SCN develop mostly postnatally. The molecular clockwork may start to be photoperiod-dependent around the 10th postnatal day.

A putative transcription factor with seven zinc-finger motifs identified in the developing suprachiasmatic nucleus by the differential display PCR method.

The suprachiasmatic nucleus (SCN) is a mammalian central circadian pacemaker. This nucleus develops in the last stage of fetal life and matures to make strong synaptic connections within 2 weeks of postnatal life to establish strong oscillation characteristics. To identify factors that initiate the circadian oscillation, we applied a differential display PCR method to developing SCN, and isolated a gene with seven zinc-finger motifs, Lot1, which encodes a gene that appeared at a very high level in the SCN during the early postnatal days. Lot1 mRNA first appeared at postnatal day 1 (P1) at a very high level, and the signal in the SCN continued to be very high until P10 and thereafter rapidly decreased until P20 and was expressed at a very faint level during adulthood. Lot1 mRNA expression was observed only in neurons of the dorsomedial SCN throughout the course of development. During the developmental stage, Lot1 mRNA expression shows a circadian rhythm with a peak in the day time and a trough at night time in both light-dark and constant dark conditions. These observations imply that Lot1 is the first identified putative transcription factor expressed only in the period of active synaptogenesis in the SCN, where Lot1 might play a role in establishing autonomous oscillation.

Programming of the fetal suprachiasmatic nucleus and subsequent adult rhythmicity.

The suprachiasmatic nucleus (SCN) is the site of the generation and entrainment of circadian rhythms. Similar to other structures, it develops throughout gestation but is still immature for some time after. This suggests that the SCN could be vulnerable to maternal influences, such as poor nutrition, stress and drugs, all of which can affect neuronal development. Evidence is accumulating that suggests that this is the case, with body size at birth influencing melatonin production in adult humans and maternal malnutrition, and stress affecting sleep in rodents. Interestingly, the maternal environment affects the phase of rhythms and the response of the circadian timing system to light pulses. The nature of these changes in adult rhythmicity is similar to those commonly associated with depression in humans. Thus, abnormal fetal programming might predispose adults to depressive illness.

Developmental maturation of pineal gland function: synchronized CREM inducibility and adrenergic stimulation.

The cAMP response element modulator (CREM) gene encodes multiple activators and repressors of cAMP-responsive transcription. Differential splicing generates a developmental switch in CREM function during spermatogenesis, while the use of an alternative promoter is responsible for the production of a cAMP-inducible transcriptional repressor, ICER (inducible cAMP early repressor). The ICER promoter is strongly inducible by cAMP because of the presence of four tandemly repeated cAMP response elements. Furthermore, ICER negatively autoregulates the ICER promoter activity, thus generating a feedback loop. CREM constitutes an early response gene of the cAMP pathway in several neuroendocrine cells. We have previously shown that CREM is highly expressed in the adult rat pineal gland at nighttime. Here, we show that the only additional site of rhythmic ICER expression within the photoneuroendocrine system is the lamina intercalaris. Ontogenetically, the ICER day-night switch and cAMP inducibility mature in the pineal gland at the end of the first postnatal week. Importantly, this correlates with the onset of melatonin synthesis and the establishment of functional adrenergic innervation. At this developmental phase we document a significant increase in protein kinase A levels, thus suggesting that ICER inducibility reflects a complete maturation of the cAMP-dependent signaling pathway at the nuclear level.

Distinct pharmacological mechanisms leading to c-fos gene expression in the fetal suprachiasmatic nucleus.

Maternal treatment with cocaine or a D1-dopamine receptor agonist induces c-fos gene expression in the fetal suprachiasmatic nuclei (SCN). Other treatments that induce c-fos expression in the fetal SCN include caffeine and nicotine. In the current article, the authors assessed whether these different pharmacological treatments activate c-fos expression by a common neurochemical mechanism. The results indicate the presence of at least two distinct pharmacological pathways to c-fos expression in the fetal rat SCN. Previous studies demonstrate that prenatal activation of dopamine receptors affects the developing circadian system. The present work shows that stimulant drugs influence the fetal brain through multiple transmitter systems and further suggests that there may be multiple pathways leading to entrainment of the fetal biological clock.

Embryonic development and maternal regulation of murine circadian clock function.

The importance of circadian clocks in the regulation of adult physiology in mammals is well established. In contrast, the ontogenesis of the circadian system and its role in embryonic development are still poorly understood. Although there is experimental evidence that the clock machinery is present prior to birth, data on gestational clock functionality are inconsistent. Moreover, little is known about the dependence of embryonic rhythms on maternal and environmental time cues and the role of circadian oscillations for embryonic development. The aim of this study was to test if fetal mouse tissues from early embryonic stages are capable of expressing endogenous, self-sustained circadian rhythms and their contribution to embryogenesis. Starting on embryonic day 13, we collected precursor tissues for suprachiasmatic nucleus (SCN), liver and kidney from embryos carrying the circadian reporter gene Per2::Luc and investigated rhythmicity and circadian traits of these tissues ex vivo. We found that even before the respective organs were fully developed, embryonic tissues were capable of expressing circadian rhythms. Period and amplitude of which were determined very early during development and phases of liver and kidney explants are not influenced by tissue preparation, whereas SCN explants phasing is strongly dependent on preparation time. Embryonic circadian rhythms also developed in the absence of maternal and environmental time signals. Morphological and histological comparison of offspring from matings of Clock-Δ19 mutant and wild-type mice revealed that both fetal and maternal clocks have distinct roles in embryogenesis. While genetic disruptions of maternal and embryonic clock function leads to increased fetal fat depots, abnormal ossification and organ development, Clock gene mutant newborns from mothers with a functional clock showed a larger body size compared to wild-type littermates. These data may contribute to the understanding of the ontogenesis of circadian clocks and the risk of disturbed maternal or embryonic circadian rhythms for embryonic development.

Melatonin administered during the fetal stage affects circadian clock in the suprachiasmatic nucleus but not in the liver.

The mammalian circadian system develops gradually during ontogenesis, and after birth, the system is already set to a phase of the mothers. The role of maternal melatonin in the entrainment of fetal circadian clocks has been suggested, but direct evidence is lacking. In our study, intact or pinealectomized pregnant rats were exposed to constant light (LL) throughout pregnancy to suppress the endogenous melatonin and behavioral rhythms. During the last 5 days of gestation, the rats were injected with melatonin or vehicle or were left untreated. After delivery, daily expression profiles of c-fos and Avp in the suprachiasmatic nuclei (SCN), and Per1, Per2, Rev-erbα, and Bmal1 in the liver were measured in 1-day-old pups. Due to the LL exposure, no gene expression rhythms were detected in the SCN of untreated pregnant rats or in the SCN and liver of the pups. The administration of melatonin to pregnant rats entrained the pups' gene expression profiles in the SCN, but not in the liver. Melatonin did not affect the maternal behavior during pregnancy. Vehicle injections also synchronized the gene expression in the SCN but not in the liver. Melatonin and vehicle entrained the gene expression profiles to different phases, demonstrating that the effect of melatonin was apparently not due to the treatment procedure per se. The data demonstrate that in pregnant rats with suppressed endogenous melatonin levels, pharmacological doses of melatonin affect the fetal clock in the SCN but not in the liver.

Vasopressin messenger ribonucleic acid in supraoptic and suprachiasmatic nuclei: appearance and circadian regulation during development.

The developmental appearance and regulation of hypothalamic vasopressin (prepropressophysin) mRNA was studied using quantitated in situ hybridization techniques. Vasopressin mRNA levels in the supraoptic nuclei were reliably detected on day 16 of gestation, while mRNA in the suprachiasmatic nuclei (SCN) was detectable on day 21. These developmental patterns correlate well with the immunohistochemical appearance of prepropressophysin translation products previously reported in these nuclei. A prominent day-night rhythm of vasopressin mRNA levels was evident in the SCN on day 21 of gestation; the rhythm was also present on postnatal days 2 and 11. No such rhythm was present in the supraoptic nuclei at any developmental stage examined. These results demonstrate regulated expression of the vasopressin gene during fetal life. Vasopressin mRNA levels in the SCN are already under specific circadian control when the nuclei are morphologically immature and lacking many of the connections found in adult animals.