N-acetyltransferase Rhythm Research Articles

Homeobox genes and melatonin synthesis: regulatory roles of the cone-rod homeobox transcription factor in the rodent pineal gland.

Nocturnal synthesis of melatonin in the pineal gland is controlled by a circadian rhythm in arylalkylamine N-acetyltransferase (AANAT) enzyme activity. In the rodent, Aanat gene expression displays a marked circadian rhythm; release of norepinephrine in the gland at night causes a cAMP-based induction of Aanat transcription. However, additional transcriptional control mechanisms exist. Homeobox genes, which are generally known to encode transcription factors controlling developmental processes, are also expressed in the mature rodent pineal gland. Among these, the cone-rod homeobox (CRX) transcription factor is believed to control pineal-specific Aanat expression. Based on recent advances in our understanding of Crx in the rodent pineal gland, we here suggest that homeobox genes play a role in adult pineal physiology both by ensuring pineal-specific Aanat expression and by facilitating cAMP response element-based circadian melatonin production.

Melatonin synthesis in the greenfrog retina in culture: I. Modulation by the light/dark cycle, forskolin and inhibitors of protein synthesis

Melatonin is synthesized in the pineal gland and the retina of vertebrates. Retinal serotonin N-acetyltransferase (NAT) activity and melatonin show a daily rhythm with high levels during the dark phase of the photocycle. In some vertebrates, these retinal NAT and melatonin rhythms are maintained in vitro. The aim of present work is to develop an eyecup culture system for the greenfrog ( Rana perezi), suitable to analyze the mechanisms of regulation of melatonin synthesis by simultaneous determination of NAT activity and melatonin release. The R. Perezi eyecups released melatonin to the culture medium in a rhythmic manner at least over a 27-h period under photocycle conditions. Nat activity and melatonin rhythms were similar to that observed in vivo under natural environmental conditions. Rana perezi retina exhibits a pronounced photosensitivity in vitro. Forskolin increased up to 2-fold the NAT activity and 4-fold the melatonin production at any lighting conditions. The addition of the translation inhibitor, cycloheximide, to the medium reduced significantly both nocturnal NAT activity and melatonin release, suggesting that de novo protein synthesis is produced daily during darkness. Actinomycin D, a transcription inhibitor, needs a longer time of action, because pre-existing mRNA must be depleted before the inhibition of melatonin release can be observed. The eyecup culture system is highly sensitive to light and chemical factors, which makes it particularly suitable as a model for the neurochemical analysis of melatonin biosynthesis in the retina of Rana perezi.

Effect of Maternal Deprivation on N-Acetyltransferase Activity Rhythm in Blinded Rat Pups

Katoh, Y., Y. Takeuchi, K. Yamazaki and K. Takahashi. Effect of maternal deprivation on N-acetyltransferase activity rhythm in blinded rat pups. Physiol Behav 63(4) 529–535, 1998. It has been reported that the rhythms of infant rats synchronize with the mother’s rhythm until the light–dark cycle comes and has strong effects on their endogenous clocks. We found that periodic maternal deprivation (PMD) was able to cause a phase shift of serotonin N-acetyltransferase (NAT) in neonatal blinded rat pups. PMD in which contact with the mother was allowed for only 4 h caused a phase shift of NAT rhythm, irrespective of the timing of contact with the mother in a day. Acute single mother deprivation caused an excess of NAT activity for more hours than usual and contact with the mother prevented such an excessive response. Mother deprivation may act as a cold stress, since artificial warming of pups gave the same results as contact with the mother. When the pups were artificially warmed by a heater during a 1-week deprivation period, a flat 24-h pattern of NAT was observed. The mechanism causing a phase shift of NAT activity rhythm of rat pups may be complicated.

Photic entrainment of the mammalian rhythm in melatonin production.

This review summarizes studies on the photic entrainment of the circadian rhythm in the rat pineal melatonin production, namely of the rhythm in N-acetyltransferase (NAT) activity, and compares the NAT rhythm resetting with preliminary results on the resetting of an intrinsic rhythmicity in the suprachiasmatic nucleus (SCN) of the hypothalamus, namely with the entrainment of the rhythm in the light-induced c-fos gene expression. Phase delaying of the NAT rhythm after various light stimuli proceeds within 1 day with almost no transients, whereas during phase advancing of the rhythm only the morning NAT decline is phase advanced within 1 day and the evening rise phase shifts through transients. A light stimulus encompassing the middle of the night may phase delay the evening NAT rise, phase advance the morning decline, compress the rhythm waveform, and eventually lower its amplitude. Similarly, a long photoperiod compresses the NAT rhythm waveform. The magnitude of phase shifts of the NAT rhythm, as well as their direction, depends on a previous photoperiod. Phase shifts of the evening rise in c-fos gene photoinduction in the SCN and of the morning decline are similar to those of the pineal NAT rhythm after all light stimuli studied so far. The data indicate that the resetting of the rhythm in melatonin production in the rat pineal gland reflects changes in the SCN functional state and suggest that the underlying SCN pacemaking system is complex.

Melatonin entrainment of the circadian N-acetyltransferase rhythm in the newborn rat pineal gland.

In 15-day-old control and vehicle-treated rats, the evening rise of the pineal N-acetyltransferase occurred at the same time as in their mothers, whereas in 5-day-old pups, the rise occurred by 2-3 hr earlier. Four-day administration of melatonin in the late day phase advanced the N-acetyltransferase rise in 15-day-old rats as compared with the rise in the vehicle-treated animals; a slight melatonin induced phase advance in 5- and 27-day-old rats was not significant. The data indicate that the newborn rat's circadian pacemaker controlling the rhythmic N-acetyltransferase rise may be entrained by exogenous melatonin. It appears, however, that the maternal melatonin transferred via milk cannot entrain the pup's pacemaker by phase advancing it, since the N-acetyltransferase rise in the pups begins earlier or at the same time as maternal melatonin production driven by the N-acetyltransferase rhythm.

Photoperiod-dependent correlation between light-induced SCN c-fos expression and resetting of circadian phase.

In rodents, brief light pulses that shift the phase of the circadian locomotor rhythm also increase c-fos gene expression in the suprachiasmatic nucleus (SCN), site of an endogenous clock that regulates such rhythmicity. Because the magnitude of photic phase shifts varies when light pulses are applied at different time points over the course of the subjective night, we examined the degree of SCN c-fos gene expression after single 30-min light pulses were delivered at time points that spanned the early and late subjective night in rats maintained in either short (8:16-h light-dark cycle) or long (16:8-h light-dark cycle) photoperiods. The light-induced level of c-fos mRNA and the number of cells expressing immunoreactive c-Fos protein were measured in the SCN by in situ hybridization and immunohistochemistry, respectively, and compared with the magnitude of the corresponding phase shifts of the circadian rhythm of pineal N-acetyltransferase (NAT) activity. We found a robust correlation between c-fos photoinduction and NAT phase shifts, but this relationship was dependent on photoperiod. The degree of c-fos gene expression was strongly correlated with the magnitude of NAT phase advances and delays under the short photoperiod and with phase advances under the long photoperiod, but not with phase delays under the long photoperiod. The data suggest that c-fos gene expression in the SCN may be involved in the photic resetting of the pineal NAT rhythm. Under the long photoperiod, however, the magnitude of phase delays may be limited by the functional state of the circadian pacemaking system.

A role for bursa fabricii and bursin in the ontogeny of the pineal biosynthetic activity in the chicken.

The tripeptide bursin (Lys-His-Gly-NH2) is a B cell differentiation hormone derived from the bursa fabricii. The latter is a cloacal diverticulum and the site of B lymphocyte differentiation and selection in aves; also the bursa fabricii is involved in endocrine functions. Herein we demonstrate that in the chicken, the bursa fabricii and bursin are crucial to the ontogeny of both the pineal response to antigenic challenge and pineal circadian synthetic activity. In early embryonically bursectomized chickens, the plasma melatonin response to immunization by porcine thyroglobulin (Tg) was abolished. Also, the amplitudes of both plasma melatonin and pineal N-acetyltransferase (NAT) circadian rhythms were reduced by 50%, whereas the activity of hydroxyindole-O-methyltransferase (HIOMT) remained unchanged. Conversely, administration of either minute amounts (100 pg, 100 fg) or highly dilute (5 x 10(-27) g) bursin, with the exception of a highest dose (100 micrograms), to bursaless embryos induced recovery of normal antigen-induced melatonin response and normal amplitudes of melatonin and NAT rhythms. These findings establish that early in embryonic life, the bursa fabricii and its derived signal (bursin) are essential for normal development of pineal synthetic activity and underline the efficacy of very dilute bursin as an informative signal.

Ontogeny of Ocular Serotonin N-Acetyltransferase Activity Daily Rhythm in Four Anuran Species

The profile of daily ocular serotonin N-acetyltransferase (NAT) rhythm has been examined during the development of four anuran amphibians in different environments and of varying phylogenetic origins: Xenopus laevis, Discoglossus pictus, Rana perezi, and Bufo calamita. A significant day/night rhythm of ocular NAT with the acrophase at midnight was first detected at stages 43/44 and 25 in X. laevis and R. perezi, respectively. Total enzymatic activity increased with body weight throughout development, although both mean levels and amplitudes of NAT activity per milligram of protein significantly decreased; this attenuation of rhythm was more significant in postmetamorphic froglets, and the acrophase was delayed about 1 hr. There were no significant differences in daily NAT profiles before and after metamorphosis in D. pictus and B. calamita. The amplitude of the NAT rhythm was maximal (>10-fold nocturnal values) in Xenopus compared with other species (2- to 3-fold).

Serotonin N-Acetyltransferase (NAT) Activity and Melatonin Levels in the Frog Retina Are Not Correlated during the Seasonal Cycle

The seasonal pattern of daily serotonin N-acetyltransferase (NAT) and melatonin rhythms in the lateral eye of Rana perezi has been characterized to assess any seasonal relationship between enzymatic activity and melatonin production and to evaluate the photothermal responses of such rhythms. In winter there was no daily rhythm either in NAT activity or in melatonin production, although the highest values of NAT activity and lowest melatonin concentrations were measured at this time. Ocular melatonin showed a high-amplitude diurnal rhythm during the summer, whereas a low-amplitude rhythm was observed in spring and autumn. Diurnal melatonin levels were temperature-independent; however, nocturnal melatonin production significantly increased with temperature. Ocular NAT was always higher at lower than at higher temperatures (two- to threefold), both at night and during the day. As suggested for pineal melatonin production in other poikilotherm species, photoperiod controls the duration of the melatonin nocturnal rise, whereas temperature regulates the amplitude of the daily melatonin rhythm in the frog retina. The lack of correlation between daily NAT and melatonin rhythms in the frog retina may result from the high thermal sensitivity of the ocular NAT and a temperature-dependent inhibition substrate of NAT activity.

Periodic mother deprivation during the light period reversed the phase of serotonin N-acetyltransferase activity rhythm of the pineal gland in rat pups

It has been reported that nursing mother rats can postnatally entrain the circadian rhythms of blinded rat pups, such as locomotor, drinking, and corticosterone rhythms. To gain more insight in the mechanism of the postnatal entrainment of such pups' circadian rhythms, we examined the serotonin N-acetyltransferase (NAT) activity rhythm in blinded rat pups subjected to periodic mother deprivation (PMD) in which mothers were periodically deprived of their pups during either half of a day. We found that only PMD during the light period shifted the phase of NAT activity rhythm in the pups. To cause a reversal of the NAT activity rhythm, it was necessary to repeat PMD for more than 6 days. PMD for 6 h each day also shifted the phase of the blinded rat pups, but it did not reverse the NAT rhythm, even when it was repeated for 10 days. In 9-h deprivation for 10 days, however, deprivation during the first 9 h of the light period reversed the phase, although the latter 9 h failed to cause reversal of the phase. On the other hand, restricted feeding of the mother took more than 11 days to reverse the phase and a foster mother in the cross-fostering experiment failed to affect the phase of pup's rhythm, when the rhythm was determined on the 11th postnatal day. These facts indicate that PMD during the light period is a potent entrainer of the pups' circadian NAT rhythms and provide a useful method of exploring the underlying mechanism of the entrainment of the pups' rhythm by the mother.

Entrainment of the circadian rhythm in the rat pineal N-acetyltransferase activity by melatonin is photoperiod dependent.

Entraining effect of melatonin on the circadian rhythm in rat pineal N-acetyltransferase (NAT) activity was studied under various photoperiods. Melatonin administration prior to dark onset for 5 successive days phase-advanced the evening NAT rise under the light:dark (LD) cycle of either LD 10:14 or LD 8:16, but not under LD 12:12. It is assumed that under the latter regime, the end of a light period exhibited a phase-delaying effect on the NAT rise. The light exposure appeared to be a stronger Zeitgeber than melatonin itself. Data show that melatonin applied in the late light period advances the evening NAT rise under a short photoperiod only; under a longer photoperiod, the phase-advancing effect of melatonin may conflict with a phase-delaying effect of the end of a light period, and the effect of light exposure overrides that of melatonin.

Entrainment of the rat circadian clock controlling the pineal N-acetyltransferase rhythm depends on photoperiod

Entrainment of the circadian clock as a function of time when a light stimulus is presented has been studied in detail while little attention has been paid to a role photoperiod may play in the resetting. To find out whether and how photoperiod affects the entrainment, resetting of the rat circadian pacemaker by delays in the evening light offset and by advances in the morning light onset, respectively, was studied in rats maintained either under a short photoperiod, with 8 h of light and 16 h of darkness per day (LD8:16) or under a long, LD18:6 photoperiod. To assess phase shifts of the clock, the suprachiasmatic nucleus controlled rhythm in the pineal N-acetyltransferase (NAT), namely the time of the evening NAT rise and the time of the morning decline, were followed. One day after a delay in lights off, on LD8:16 the NAT rhythm with a normal amplitude was retained following longer delays of the light offset and the maximum phase delay of the NAT rise was 3 times larger than on LD18:6. One day after an advance in lights on, the NAT decline was phase advanced under both photoperiods; on LD8:16 the maximum shift was 3 times as large as on LD18:6. On LD8:16, the NAT rise was not shifted after shorter advances in lights on and became phase delayed only when the light onset was brought forward to before midnight while on LD18:6 the NAT rise was phase delayed after any, even a mere 1 h, advance in lights on. The data show that magnitude and direction of phase shifts of the NAT rhythm depend not only on the time of light presentation but on photoperiod as well. Difference in resetting of the rhythm under various photoperiods may reflect photoperiod-dependent changes of an underlying pacemaker.

Resetting of the rat circadian clock after a shift in the light/dark cycle depends on the photoperiod

Adjustment of the circadian clock to shifts in the light/dark (LD) cycle was assessed from the rat pineal N-acetyltransferase (NAT) rhythm which is controlled by a pacemaker in the suprachiasmatic nucleus of the hypothalamus. Re-entrainment to an 8-h delay in the LD cycle took more than 3 days in rats maintained under a regime with 18 h of light and 6 h of darkness per day (LD 18:6) whereas it was completed within 3 days in those maintained under LD 12:12. Re-entrainment to an advance in the LD cycle proceeded through a transient diminution or almost disappearance of the NAT rhythm amplitude following a 5-h, 3-h and even a mere 2-h advance shift under LD 18:6, whereas no such diminution occurred under LD 12:12 even after a 5-h advance shift. Altogether, the data indicate that resetting of the circadian clock after shifts in the LD cycle depends on the photoperiod.

Circadian rhythm of tryptophan hydroxylase activity in chicken retina

1. Retinal tryptophan hydroxylase activity in chickens (1-4 weeks old and embryos) was estimated by determination of levels of 5-hydroxytryptophan (5HTP) in retinas at defined intervals after inhibition of aromatic L-amino acid decarboxylase with m-hydroxybenzylhydrazine (NSD1015). 2. The relationship of tryptophan hydroxylase activity to photoperiod was explored. In chickens maintained on a 12-hr light: 12-hr dark cycle, a diurnal cycle in tryptophan hydroxylase activity was observed. Activity during middark phase was 4.4 times that seen in midlight phase. Cyclic changes in tryptophan hydroxylase activity persisted in constant darkness with a period of approximately 1 day, indicating regulation of the enzyme by a circadian oscillator. The phase of the tryptophan hydroxylase rhythm was found to be determined by the phase of the light/dark cycle. The relationship of the tryptophan hydroxylase rhythm to the light/dark cycle mirrors previously described rhythms of melatonin synthesis and serotonin N-acetyltransferase (NAT) activity in the retina. 3. Light exposure for 1 hr during dark phase suppressed NAT activity by 82%, while tryptophan hydroxylase activity was suppressed by only 30%. 4. Based on the differential responses of retinal NAT activity and tryptophan hydroxylase activity to acute light exposure during dark phase, it was predicted that exposure to light during dark phase would divert serotonin in the retina from melatonin biosynthesis to oxidation by MAO. In support of this, levels of 5-hydroxyindole acetic acid (5HIAA) in retina were found to be elevated approximately two-fold in chickens exposed to 30 min of light during dark phase. In pargyline-treated chickens, 2 hr of light exposure during dark phase was found to increase retinal serotonin levels by 64% over pargyline-treated controls. 5. Cyclic changes in tryptophan hydroxylase activity and NAT activity persisted for 2-3 days in constant light. Tryptophan hydroxylase activity at mid-night gradually decreased on successive days in constant light; on the first day of constant light, tryptophan hydroxylase activity at mid-night was 70% of activity seen during middark phase of the normal light/dark cycle and decreased further on subsequent days. In contrast, on each of 3 days of constant light, NAT activity at mid-night was approximately 15% of normal middark phase activity. 6. Cycloheximide completely inhibited the nocturnal increase in tryptophan hydroxylase activity when given immediately before light offset. The nocturnal increase in NAT activity was inhibited in a similar fashion. 7. Like the development of the NAT rhythm, cyclic changes of tryptophan hydroxylase activity in the retinas of chickens began on or immediately before the day of hatching. hatching.(ABSTRACT TRUNCATED AT 400 WORDS)

Rate of re-entrainment of circadian rhythms to advances of light-dark cycles may depend on ways of shifting the cycles

When an 8-h advance of a light-dark (LD) cycle was accomplished by shortening of one dark period by 8 h, the rat pineal N-acetyltransferase rhythm was abolished during the first 3 subsequent cycles and reappeared in its original waveform during the fifth cycle only. When the 8-h advance shift was accomplished by lengthening of two consecutive light periods by 8 h, the N-acetyltransferase rhythm persisted and attained its original waveform by 2 days earlier than under the former shift. Re-entrainment is thus more rapid when the advance shift is accomplished by twice delaying than by once advancing of the LD cycle by 8 h.

The rat pineal N-acetyltransferase rhythm persists after a five-hour, but disappears temporarily after a seven-hour advance of the light-dark cycle: A six-hour shift may be a turning point

After a 5-h advance of a lighting regime with 12 h of light and 12 h of darkness by shortening of one dark period, the rat pineal N-acetyltransferase (NAT) rhythm was retained, though at the beginning in a changed waveform, and re-entrained to the shift within 5 to 6 cycles. After a 7-h advance of the regime, the NAT rhythm faded away between the 2nd and 4th cycle and re-entrained to the shift within 7 cycles. A 6-h advance of the lighting regime might thus be a turning point for the persistence or temporal disappearance of the NAT rhythm.

Melatonin and porphyrin in the harderian glands of the syrian hamster: Orcadian patterns and response to autumnal conditions

1. 1. Adult male Syrian hamsters were killed at nine intervals during a 24 hr period in the autumn, after 2 months either indoors in controlled conditions or in natural outdoor conditions. 2. 2. Harderian glands were taken for determination of N-acetyltransferase (NAT) and hydroxyindole- O-methyltransferase (HIOMT) activities and melatonin and porphyrin concentrations. 3. 3. Mean 24 hr Harderian NAT and melatonin values were lower outside than inside. 4. 4. Twenty-four hour melatonin rhythms were detected with similar daytime (afternoon) acrophases in both environmental conditions. 5. 5. An NAT rhythm was seen only in animals kept inside, with a circadian maximum in the late dark phase. 6. 6. Mean 24 hr HIOMT activity was slightly higher outdoors than indoors, and 24 hr rhythms were not detected in either condition. 7. 7. Mean porphyrin concentrations were higher outdoors, with 24 hr rhythms detected in both conditions and a significantly earlier nocturnal circadian maximum outdoors.

Melatonin Entrains the Circadian Rhythm in the Rat Pineal N-Acetyltransferase Activity

It has been recently suggested that in mammals the pineal hormone melatonin may be involved not only in transduction of a photoperiodic information, but in a subtle modulation of a phase of the circadian system as well. This suggestion has been based mainly on data about entrainment of the locomotor activity rhythm by melatonin. The present study was undertaken to check whether melatonin may phase-shift also other circadian rhythms, namely the rhythm in pineal N-acetyltransferase activity which is supposed to drive the rhythmic melatonin production in the rat. 50-day-old male Wistar rats were maintained on a regimen of 10 h of light and 14 h of darkness per day. Vehicle or melatonin (1mg/kg rat weight) was injected subcutaneously just before the dark onset. After a single melatonin injection or after administration of melatonin for 5 successive days or after a 4-day treatment with melatonin and a 1-day withdrawal, the evening N-acetyltransferase rise was phase-advanced relative to the rise in rats treated with vehicle only; the phase shift was larger after a repeated than after a single melatonin injection. Under all above-mentioned paradigms of melatonin administration, the morning N-acetyltransferase decline was less phase-advanced than the evening rise. Persistence of the phase advance of the N-acetyltransferase rise after withdrawal of melatonin treatment and a larger phase shift after a repeated than after a single melatonin administration suggest that melatonin may entrain directly a circadian pacemaker controlling the N-acetyltransferase rhythm and affect thus its own rhythmic production.

Melatonin accelerates reentrainment of the circadian rhythm of its own production after an 8-h advance of the light-dark cycle.

The rhythm in melatonin production in the rat is driven by a circadian rhythm in the pineal N-acetyltransferase (NAT) activity. Rats adapted to an artificial lighting regime of 12 h of light and 12 h of darkness per day were exposed to an 8-h advance of the light-dark regime accomplished by the shortening of one dark period; the effect of melatonin, triazolam and fluoxetine, together with 5-hydroxytryptophan, on the reentrainment of the NAT rhythm was studied. In control rats, the NAT rhythm was abolished during the first 3 cycles following the advance shift. It reappeared during the 4th cycle; however, the phase relationship between the evening rise in activity and the morning decline was still compressed. Melatonin accelerated the NAT rhythm reentrainment. In rats treated chronically with melatonin at the new dark onset, the rhythm had already reappeared during the 3rd cycle, in the middle of the advanced night, and during the 4th cycle, the phase relationship between the evening onset and the morning decline of the NAT activity was the same as before the advance shift. In rats treated chronically with melatonin at the old dark onset or in those treated with melatonin 8 h, 5 h and 2 h after the new dark onset during the 1st, 2nd and 3rd cycle, respectively, following the advance shift, the NAT rhythm reappeared during the 3rd cycle as well but in the last third of the advanced night only. Neither triazolam nor fluoxetine together with 5-hydroxytryptophan administered around the new dark onset facilitated NAT rhythm reentrainment after the 8-h advance of the light-dark cycle.

Mechanism of the re-entrainment of the circadian rhythm in the rat pineal N-acetyltransferase activity to an eight-hour advance of the light-dark cycle: phase-jump is involved

During the re-entrainment of the rat pineal N-acetyltransferase (NAT) rhythm to an 8-h advance of a light-dark (LD) cycle accomplished by shortening of one dark period, the advanced light period phase-delayed the evening NAT rise and phase-advanced the morning decline. The phase-relationship between the rise and the decline became so compressed that after the second and the third light period the NAT rhythm was not expressed even in constant darkness and reappeared only within another cycle at the time of the original night. Only after the fourthdvanced light period, the NAT rhythm phase-jumped into the advanced night.