From the Pineal Gland to the Central Clock in the Brain: Beginning of Studies of the Mammalian Biological Rhythms in the Institute of Physiology of the Czech Academy of Sciences.

The Institute of Physiology of the Czech Academy of Sciences (CAS) has been involved in the field of chronobiology, i.e., in research on temporal regulation of physiological processes, since 1970. The review describes the first 35 years of the research mostly on the effect of light and daylength, i.e., photoperiod, on entrainment or resetting of the pineal rhythm in melatonin production and of intrinsic rhythms in the central biological clock. This clock controls pineal and other circadian rhythms and is located in the suprachiasmatic nuclei (SCN) of the hypothalamus. During the early chronobiological research, many original findings have been reported, e.g. on mechanisms of resetting of the pineal rhythm in melatonin production by short light pulses or by long exposures of animals to light at night, on modulation of the nocturnal melatonin production by the photoperiod or on the presence of high affinity melatonin binding sites in the SCN. The first evidence was given that the photoperiod modulates functional properties of the SCN and hence the SCN not only controls the daily programme of the organism but it may serve also as a calendar measuring the time of a year. During all the years, the chronobiological community has started to talk about "the Czech school of chronobiology". At present, the today´s Laboratory of Biological Rhythms of the Institute of Physiology CAS continues in the chronobiological research and the studies have been extended to the entire circadian timekeeping system in mammals with focus on its ontogenesis, entrainment mechanisms and circadian regulation of physiological functions. Key words: Pineal, Melatonin, AA-NAT rhythm, Light entrainment, Photoperiod, SCN clock.

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.

Ontogenesis of photoperiodic entrainment of the molecular core clockwork in the rat suprachiasmatic nucleus.

The molecular mechanism underlying a generation of circadian rhythmicity within the suprachiasmatic nucleus (SCN) is based on interactive negative and positive feedback loops that drive the rhythmic transcription of clock genes and translation of their protein products. In adults, the molecular mechanism is affected by seasonal changes in day length, i.e., photoperiod. The photoperiod modulates phase, waveform, and amplitude of the rhythmic clock genes expression as well as the phase relationship between their profiles. To ascertain when and how the photoperiod affects the circadian core clock mechanism during ontogenesis, the rhythmic expression of clock genes, namely of Per1, Per2, Cry1 and Bmal1 was determined in 3-, 10- and 20-day-old rat pups maintained under either a long photoperiod with 16 h of light and 8 h of darkness per day (LD 16:8) or under a short, LD 8:16 photoperiod. The daily profiles in the level of clock genes mRNA were studied in constant darkness. The photoperiod affected the profile of Per1 and Per2 mRNA in 20- and 10- but not yet in 3-day-old pups. Expression of Cry1 was affected only in 20-day-old pups, whereas expression of Bmal1 was not yet affected even in 20-day-old rats. The results demonstrate no effect of the photoperiod on 3-day-old pups, only partial entrainment of the molecular core clockwork in 10-day-old pups and a more mature, though not yet fully complete, entrainment in 20-day-old pups as compared with adult animals. The developmental interval when the photoperiod begins to entrain the core clock mechanism completely might thus occur around the time of weaning.

Dynamics of discrete entrainment of the circadian rhythm in the rat pineal N-acetyltransferase activity during transient cycles.

To elucidate entrainment of a pacemaker controlling the N-acetyltransferase (NAT) rhythm in the rat pineal gland, we studied the phase response curves (PRCs) of this rhythm. We exposed 50- to 60-day-old male Wistar rats maintained in a light-dark cycle (LD 12:12) to a 1-min light pulse at different times before midnight or at various times throughout the whole night. We then released them into constant darkness and studied the morning NAT decline during the night when rats were pulsed before midnight, as well as the evening NAT rise and the morning decline after 4 days following the pulses. The PRC for the first NAT decline and the PRCs for the NAT rise and decline after 4 days were compared with published transient PRCs (Illnerová and Vanecek, 1982b), in order to obtain a complete picture of the dynamics of the NAT rhythm entrainment during the transient cycles. Phase delays in the NAT rise due to a pulse before midnight were complete (i.e., identical to those of day 4) on day 1. Phase delays in the NAT decline were almost complete on day 1, while incomplete phase delays were observed on day 0. Phase advances in the NAT rise and decline due to a pulse past midnight had different dynamics: Advances in the decline were complete on day 1, while advances in the rise were absent on day 1 and much smaller than in the decline on day 4. The results are discussed in terms of a two-component (E-M) pacemaker controlling the NAT rhythm. The NAT rise may reflect the phase of the E-component, while the decline reflects the M-component. Phase delays of the E-component are accomplished within one cycle, and so are phase advances of the M-component. However, although delays of E already result in delays of M one cycle after the pulse, it takes several transient cycles before advances of M begin to induce advances of E.

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.

Different mechanisms of phase delays and phase advances of the circadian rhythm in rat pineal N-acetyltransferase activity.

The circadian rhythm in rat pineal N-acetyltransferase (NAT) activity, which drives the rhythm in melatonin production, is controlled by a pacemaker located in the suprachiasmatic nucleus of the hypothalamus. As the NAT rhythm has two well-defined phase markers--namely, the time of the evening activity rise and of the morning decline--it is suitable for studies of the entrainment of the pacemaker by environmental light. Phase delays of the NAT rhythm proceed more rapidly than phase advances. One day after a brief light pulse applied before midnight, or after a delay in evening lights-off, or a delay of a light-dark (LD) cycle, phase delays of the evening NAT rise result in almost corresponding delays of the morning NAT decline. Consequently, the NAT rhythm is phase-shifted, but its pattern does not change. One day after a brief light pulse applied past midnight, or after bringing forward morning lights-on, or after an advance of an LD cycle, the morning NAT decline is phase-advanced, but the evening rise is not phase-advanced at all or may even by phase-delayed. Consequently, the phase relationship between the evening NAT activity onset and the morning offset may be compressed considerably, and it may take several transient cycles before phase advances of the morning NAT decline are followed by corresponding advances of the evening NAT rise. Due to the phase-delaying effect of evening light on the NAT rise and to the phase-advancing effect of morning light on the NAT decline, the phase relationship between the NAT rise and the decline is compressed on long days and decompressed on short days. Different phase shifts of the evening NAT rise and of the morning decline, even in opposite directions, are consistent with the hypothesis of a complex, two-component (evening-morning, or E-M) pacemaker controlling the NAT rhythm. As the E-M phase relationship determines duration of the high night melatonin production, and the duration of the nocturnal melatonin pulse may convey information on daylength, the data are consistent with the internal coincidence model for photoperiodic time measurement.

Encoding le quattro stagioni within the mammalian brain: photoperiodic orchestration through the suprachiasmatic nucleus.

Within the suprachiasmatic nucleus (SCN) is a pacemaker that not only drives circadian rhythmicity but also directs the circadian organization of photoperiodic (seasonal) timekeeping. Recent evidence using electrophysiological, molecular, and genetic tools now strongly supports this conclusion. Important questions remain regarding the SCN's precise role(s) in the brain's photoperiodic circuits, especially among different species, and the cellular and molecular mechanisms for its photoperiodic "memory." New data suggesting that SCN "clock" genes may also function as "calendar" genes are a first step toward understanding how a photoperiodic clock is built from cycling molecules.

Assembling a clock for all seasons: are there M and E oscillators in the genes?

The hypothesis is advanced that the circadian pacemaker in the mammalian suprachiasmatic nucleus (SCN) is composed at the molecular level of a nonredundant double complex of circadian genes (per1, cry1, and per2, cry2). Each one of these sets would be sufficient for the maintenance of endogenous rhythmicity and thus constitute an oscillator. Each would have slightly different temporal dynamics and light responses. The per1/cry1 oscillator is accelerated by light and decelerated by darkness and thereby tracks dawn when day length changes. The per2 /cry2 oscillator is decelerated by light and accelerated by darkness and thereby tracks dusk. These M (morning) and E (evening) oscillators would give rise to the SCN's neuronal activity in an M and an E component. Suppression of behavioral activity by SCN activity in nocturnal mammals would give rise to adaptive tuning of the endogenous behavioral program to day length. The proposition-which is a specification of Pittendrigh and Daan's E-M oscillator model-yields specific nonintuitive predictions amenable to experimental testing in animals with mutations of circadian genes.

Melatonin rhythm in human milk.

The pineal hormone melatonin exhibits a circadian rhythm in body fluids. No data are available on melatonin in human milk. The present study was undertaken to determine whether melatonin is detectable in human milk and, if so, whether it exhibits a daily rhythm. Blood and milk were sampled between 1400-1700 h and again between 0200-0400 h from 10 mothers 3-4 days after delivery. Melatonin in both fluids was beyond the limit of detection during the day, whereas during the night, its concentration was 280 +/- 34 pmol/L in serum and 99 +/- 26 pmol/L in milk. Six mothers collected milk after each feeding throughout 1 24-h period within 3 months after delivery. Melatonin in the milk of all subjects exhibited a pronounced daily rhythm, with high levels during the night and undetectable levels during the day. The presence of the rhythm in milk suggests that melatonin fluctuations in milk might communicate time of day information to breast-fed infants.

Human hydroxyindole-O-methyltransferase: presence of LINE-1 fragment in a cDNA clone and pineal mRNA.

Hydroxyindole-O-methyltransferase (HIOMT) catalyzes the last step in the synthesis of the pineal hormone melatonin. In this study, an HIOMT clone was isolated from a human pineal cDNA library using synthetic oligonucleotide probes based on the bovine HIOMT sequence. The human sequence is unusual because it contains a 3' fragment (84 bp) of the LINE-1 sequence, a highly repetitive sequence in the human genome and the genome of some primates and rodents. Exclusive of this LINE-1 fragment, the human HIOMT clone is 75% and 63% homologous to bovine and avian HIOMT sequences, respectively. The deduced amino acid sequence of the human cDNA clone encodes a 41.6-kD protein. In addition, the sequence is 70% and 57% identical and 81% and 73% similar to bovine and avian HIOMT, respectively. In agreement with the results of earlier studies, it was found that vertebrate HIOMT amino acid sequences are not homologous to any other vertebrate proteins, including several methyltransferases. However, HIOMT exhibits homology with a plant O-methyltransferase and an internal 120-amino-acid region is approximately 35% identical to a region of four bacterial O-methyltransferases. The results of PCR and Southern blot analysis indicate that three species of HIOMT mRNA are typically present in the human pineal gland, only one of which contains the LINE-1 fragment. An antiserum was raised against a mixture of three synthetic peptides, corresponding to three regions of the deduced amino acid sequence of human HIOMT. This antiserum detected a single immunoreactive protein in Western blot analysis of human pineal glands. The size of the protein (approximately 42 kD) is identical to that predicted from the HIOMT clone, including the LINE-1 fragment. The human HIOMT sequence should be useful in further studies of this enzyme and will also be of special importance in evaluating the functional significance of the inclusion of a fragment of the LINE-1 in an mRNA.

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.

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 fourth advanced light period, the NAT rhythm phase-jumped into the advanced night.

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.

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 (LD 8:16) or under a long, LD 18:6 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 LD 8:16 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 LD 18:6. One day after an advance in lights on, the NAT decline was phase advanced under both photoperiods; on LD 8:16 the maximum shift was 3 times as large as on LD 18:6. On LD 8:16, 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 LD 18:6 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.

Endogenous melatonin signal does not mediate the effect of photoperiod on the rat suprachiasmatic nucleus.

The duration of the nocturnal interval during which light can induce high Fos-like immunoreactivity in the suprachiasmatic nucleus is a function of the photoperiod to which rats are exposed. The effect of photoperiod is not altered by pinealectomy indicating that day length affects the functional state of the suprachiasmatic nucleus circadian pacemaking system in the rat directly, not via the pineal melatonin signal.

The circadian rhythm in plasma melatonin concentration of the urbanized man: the effect of summer and winter time.

Plasma melatonin rhythms were measured in healthy urbanized persons between July 4 and 5 and between January 13 and 14. In contrast to findings in other mammals studied thus far, no difference in the duration of elevated night melatonin concentration was observed between summer and winter. In winter, melatonin rhythms were phase-delayed by about 1.5 h as compared with summer patterns.

Adjustment of the rat pineal N-acetyltransferase rhythm to change from long to short photoperiod depends on the direction of the extension of the dark period.

Extension of the rat pineal N-acetyltransferase rhythm after transition from LD 16:8 to LD 8:16 proceeded more rapidly when the transition was accomplished by prolongation of the dark period into the morning than into the evening hours. After symmetrical or evening prolongation of the dark period, adjustment of the rhythm to the LD 8:16 regimen was almost completed within 6 days. After prolongation of darkness into the morning hours, a temporal overextension of the rhythm occurred.

Adjustment of the rat pineal N-acetyltransferase rhythm to eight-hour shifts of the light-dark cycle: advance of the cycle disturbs the rhythm more than delay.

After an 8-h delay of a light-dark (LD) cycle by lengthening of one light period, the rat pineal N-acetyltransferase (NAT) rhythm adjusted to the delay shift almost within one cycle. After an 8-h advance of the LD cycle by shortening of one dark period, the NAT rhythm adjusted to the advance shift within 5 cycles only; during the first 2-3 cycles the rhythm was abolished.

Hypothalamic melatonin receptor sites revealed by autoradiography.

125I-Melatonin was used to localize and characterize the melatonin receptor sites in the rat hypothalamus. Autoradiography revealed that displaceable 125I-melatonin binding occurred in suprachiasmatic nuclei and median eminence only. Further studies performed on crude membrane fractions from median eminences revealed high affinity (Kd = 21 pM) melatonin binding sites (Bmax = 8.5 fmol/mg protein). The order of potency of various indole amines to inhibit 125I-melatonin binding was melatonin much greater than N-acetyl-5-hydroxytryptamine greater than 5-methoxytryptamine greater than 5-hydroxytryptamine.

Exposure to long summer days affects the human melatonin and cortisol rhythms.

Exposure of 8 human subjects in summer to a natural 16 h bright light photoperiod phase advanced the morning salivary melatonin decline and cortisol rise and shortened the nocturnal melatonin signal by 2 h relative to the winter patterns of the same subjects followed under a combined artificial and natural light 16 h photoperiod. The data suggest that summer days experienced from sunrise till sunset and not winter days with a combined artificial and natural light long photoperiod evoke a true long day response of the human circadian system.