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.

Seasonal molecular timekeeping within the rat circadian clock.

In temperate zones duration of daylight, i.e. photoperiod, changes with the seasons. The changing photoperiod affects animal as well as human physiology. All mammals exhibit circadian rhythms and a circadian clock controlling the rhythms is located in the suprachiasmatic nucleus (SCN) of the hypothalamus. The SCN consists of two parts differing morphologically and functionally, namely of the ventrolateral (VL) and the dorsomedial (DM). Many aspects of SCN-driven rhythmicity are affected by the photoperiod. The aim of the present overview is to summarize data about the effect of the photoperiod on the molecular timekeeping mechanism in the rat SCN, especially the effect on core clock genes, clock-controlled genes and clock-related genes expression. The summarized data indicate that the photoperiod affects i) clock-driven rhythm in photoinduction of c-fos gene and its protein product within the VL SCN, ii) clock-driven spontaneous rhythms in clock-controlled, i.e. arginine-vasopressin, and in clock-related, i.e. c-fos, gene expression within the DM SCN, and iii) the core clockwork mechanism within the rat SCN. Hence, the whole central timekeeping mechanism within the rat circadian clock measures not only the daytime but also the time of the year, i.e. the actual season.

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.

Spontaneous c-Fos rhythm in the rat suprachiasmatic nucleus: location and effect of photoperiod.

A recently reported circadian rhythm in the spontaneous c-Fos immunoreactivity in the rat suprachiasmatic nucleus (SCN) is expressed mostly in the dorsomedial (dm) SCN, where vasopressinergic cells are located. The aim of the present study is to find out whether day length, i.e., photoperiod, affects the dm-SCN rhythm and, if so, how the rhythm adjusts to a change from a long to a short photoperiod. In addition, a question of whether the spontaneous c-Fos production is localized in vasopressin- producing cells or in other cells is also studied to characterize further the dm-SCN rhythmicity. Combined immunostaining for c-Fos and arginine vasopressin (AVP) revealed that most of c-Fos immunopositive cells were devoid of AVP; the results suggest that c-Fos-producing cells in the dm-SCN are mostly not identical with those producing AVP. In rats maintained under a long photoperiod with 16:8-h light-dark cycle (LD 16:8) daily and then released into darkness, the time of the afternoon and evening decline of the spontaneous c-Fos immunoreactivity in the dm-SCN differed just slightly from the time in rats maintained originally under a short LD 8:16 photoperiod; however, the morning c-Fos rise occurred about 4 h earlier under the long than under the short photoperiod. After a change from a long to a short photoperiod, a rough but not yet a fine adjustment of the morning c-Fos rise to the change was accomplished within 3-6 days. The results show that similar to the recently reported ventrolateral SCN rhythmicity, the intrinsic dm-SCN rhythmicity is also affected by the photoperiod and suggest that the whole SCN state is photoperiod dependent.

c-Fos rhythm in subdivisions of the rat suprachiasmatic nucleus under artificial and natural photoperiods.

Recent studies have shown that the waveform of the rhythm of c-Fos photoinduction in the ventrolateral (vl) part of the suprachiasmatic nucleus (SCN) and that of the rhythm in the spontaneous c-Fos production in the dorsomedial (dm) part of the SCN in rats released into constant darkness depend on the photoperiod under which the animals were previously maintained. The aim of the present study was to find out how the rhythms of c-Fos immunoreactivity in both SCN subdivisions are affected by actual light-dark (LD) cycles with various photoperiods, either artificial or natural ones, that animals may usually experience. Rats were maintained under artificial LD cycles, with either a long (16-h photoperiod) or a short (8-h photoperiod) or under natural daylight. In the latter case, c-Fos rhythms were followed in the summer when the photoperiod lasted about 16 h or in winter when it lasted only 8 h. The rhythms of c-Fos immunoreactivity under natural daylight did not differ significantly from those under corresponding artificial photoperiods. Under a long photoperiod, the morning c-Fos rise in the dm- as well as in the vl-SCN occurred about 4 h earlier than under a short one. In both SCN subdivisions, the interval when the nighttime c-Fos immunoreactivity was low, was shorter under a long than under a short photoperiod by roughly 6 h. The morning c-Fos rise in the dm-SCN always preceded that in the vl-SCN. Whereas in the former one the rise was due to the endogenous dm-SCN rhythmicity, in the latter one the rise was induced by the morning light onset. The results show that whereas c-Fos rhythmicity under actual LD cycles is affected by the photoperiod in both SCN subdivisions, mechanism of c-Fos induction in the dm-SCN differs from that in the vl-SCN.

Hormones, subjective night and season of the year.

Production and release of many mammalian hormones exhibit circadian rhythms controlled by a pacemaker located in the suprachiasmatic nuclei (SCN) of the hypothalamus. Under conditions when the circadian pacemaker free-runs with a period close to, but not equal to 24 h, subjective day and night may not be identical with the environmental day and night. The present study was aimed to define the phase and state of the circadian pacemaker when the circadian system is experiencing subjective night and to ascertain whether and how such a defined subjective night depends on the photoperiod. The results indicate that the subjective night may be defined as the time interval when i) light stimuli can reset the circadian system, ii) pineal melatonin production and photic induction of the c-Fos gene in the ventrolateral SCN are high, and iii) the spontaneous c-Fos protein production in the dorsomedial SCN is low. Such a defined subjective night and, logically, the whole circadian pacemaking system depend on the photoperiod and hence on the season of the year which the animals are experiencing.

Gate for photic resetting of intrinsic rhythmicity of the rat suprachiasmatic nucleus under a long photoperiod.

In rats maintained under a long photoperiod and then released into darkness, the time interval enabling photic resetting of the intrinsic rhythmicity of the suprachiasmatic nucleus (SCN), namely of the rhythm in the light-induced c-Fos production, was similar to the previously reported time interval enabling high c-fos photoinduction in the SCN (Sumová, A., Trávnícková, Z., Peters, R., Schwartz, W.J. and Illnerová, H., The rat suprachiasmatic nucleus is a clock for all seasons. Proc. Natl. Acad. Sci. USA, 92 (1995) 7754-7758). The data indicate that both intervals may represent the same window for the photic sensitivity of the SCN pacemaking program.

Daily profiles of arginine vasopressin mRNA in the suprachiasmatic, supraoptic and paraventricular nuclei of the rat hypothalamus under various photoperiods.

Daily rhythm of arginine vasopressin (AVP) mRNA levels in the suprachiasmatic nucleus (SCN) of rats maintained under a short, LD 8:16 photoperiod differed from that of rats maintained under a long, LD 16:8 photoperiod: under the short photoperiod the morning AVP rise occurred significantly later than under the long one. Daily profiles of AVP mRNA in the supraoptic and paraventricular nuclei were not rhythmic and AVP mRNA levels under LD 8:16 did not differ from those under LD 16:8. The data indicate that photoperiod affects selectively the clock driven AVP gene expression in the SCN.

[Sleep disorders and the 24-hour profile of melatonin and cortisol]. / Poruchy spánku a 24 hodinový profil melatoninu a kortizolu.

The aim of our study was to assess the prevalence of the specific disorders of the circadian rhythm of cortisol and melatonin in the patients with sleep disorders. The group of our patients consisted of 93 persons (25 men, 68 women), from 4-72 years (mean age 38.3 years) with a sleep disorders. These patients were studied on Department of Neurology of 1st Medical Faculty of Charles University in Prague during years 1997-1999. Patients were divided by the clinical diagnosis into many subgroups: idiopathic hypersomnia (IH) 24 patients and other hypersomnias 8, narcolepsy 22, degenerative disorders 9, delay sleep phase syndrome (DSPS) 7, periodic leg movements syndrome (PLMS) 6, insomnia 7 and Parkinson's disease 10 patients. Twelve salivary samples were taken from each patient during a period of 24 hours in order to investigate the circadian secretion pattern of melatonin and cortisol. Salivary melatonin and cortisol levels were estimated by radioimmunoassay in the Department of Physiology of the Academy of Science, Czech Republic. Significant differences were found between our patients and the control group in idiopathic hypersomnia--acrophase of melatonin was delayed and secretion was prolonged. Patients suffered from narcolepsy often displayed multiple peak of melatonin secretion. The peak of the melatonin concentration occurred later in DSPS (non significantly). Low level of melatonin and prolonged signal of melatonin was in Parkinson's disease patients.

Adjustment of the human circadian system to changes of the sleep schedule under dim light at home.

In a field study at home, eight volunteers experienced a sleep period from 01:00 to 09:00 h encompassed by dim light for 6 days, then for another 6 days they were subjected to a 3-h advance of the sleep period, and for the last 6 days they were subjected to a 3-h delay of the sleep period, i.e. they experienced again the original sleep schedule. Following the 3-h advance of the sleep period, the circadian salivary melatonin and cortisol rhythms phase advanced by about 1 h within 6 days as compared with the original rhythm profiles, following the subsequent 3-h delay of the sleep period, both rhythms phase delayed by about 1 h within 6 days and returned roughly to their original phase. The data indicate that shifting of the sleep time under dim light at home, which may occur commonly in everyday life, phase shifts the human circadian system.

Adjustment of the human melatonin and cortisol rhythms to shortening of the natural summer photoperiod.

Fifteen human subjects were exposed to natural outdoor summer light from 0415 h until 2000 h for 4 days and then from 0800 h until 1600 h for another 4 days. Following shortening of the natural summer photoperiod, times of the morning salivary melatonin decline and cortisol rise did not change whereas the time of the evening melatonin rise phase advanced by about 1.5 h within 1 day and further did not change significantly. Consequently, the melatonin signal duration extended markedly within 1 day. The data show that the compressed melatonin rhythm waveform in humans experiencing a long natural summer photoperiod from sunrise until sunset may change rapidly following a shortening of the photoperiod.

Spontaneous rhythm in c-Fos immunoreactivity in the dorsomedial part of the rat suprachiasmatic nucleus.

In rats maintained for 2 days in constant darkness, the suprachiasmatic nucleus exhibited a circadian rhythm in c-Fos immunoreactivity, with the maximum in the morning and trough during the subjective night. In contrast to the night-time photic c-Fos induction occurring in the ventrolateral part of the nucleus, the spontaneous rhythmic c-Fos induction in darkness occurred in the dorsomedial part and might indicate an elevated dorsomedial neuronal activity in the early subjective day.

Photic resetting of intrinsic rhythmicity of the rat suprachiasmatic nucleus under various photoperiods.

To date, photic entrainment of the mammalian circadian system has been studied by following phase shifts of overt rhythms in the periphery governed by a circadian pacemaker located in the suprachiasmatic nucleus (SCN). The present study follows for the first time photic resetting of intrinsic rhythmicity of the SCN itself. Rats maintained under either a shorter photoperiod, with 12 h of light and 12 h of darkness per day, or under a long, 18:6-h light-dark photoperiod were exposed to a light stimulus during the dark period and then released into darkness, and the next day the SCN rhythm in the light-stimulated c-Fos protein immunoreactivity was followed as a marker of the SCN endogenous rhythmicity. After a light stimulus in the early night, the evening rise in the photic elevation of Fos protein photoinduction as well as the morning decline were phase delayed within one cycle. After a light stimulus in the late night, only the morning decline in the photic elevation of Fos was phase advanced the next night, not the evening rise; consequently, the interval enabling high photic elevation of Fos was reduced. After a light stimulus was administered around the middle of the night, the next night the evening rise in the light-stimulated Fos was eventually phase delayed, the morning decline was phase advanced, and the rhythm amplitude was reduced significantly; under 18:6-h light-dark, a mere 5-min light exposure exhibited such effects. The data indicate that resetting of the SCN rhythmicity in the light-elevated c-Fos 1 day after a resetting stimulus administration, i.e., during transient cycles, may proceed via nonparallel phase shifts of the evening rise and of the morning decline of the light-stimulated Fos, and via amplitude lowering and suggest a complex circadian pacemaking system in the rat SCN.

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.