Antoni Díez-Noguera

Forced Desynchronization of Dual Circadian Oscillators within the Rat Suprachiasmatic Nucleus

The circadian clock in the suprachiasmatic nucleus of the hypothalamus (SCN) contains multiple autonomous single-cell circadian oscillators and their basic intracellular oscillatory mechanism is beginning to be identified. Less well understood is how individual SCN cells create an integrated tissue pacemaker that produces a coherent read-out to the rest of the organism. Intercellular coupling mechanisms must coordinate individual cellular periods to generate the averaged, genotype-specific circadian period of whole animals. To noninvasively dissociate this circadian oscillatory network in vivo, we (T.C. and A.D.-N.) have developed an experimental paradigm that exposes animals to exotic light-dark (LD) cycles with periods close to the limits of circadian entrainment. If individual oscillators with different periods are loosely coupled within the network, perhaps some of them would be synchronized to the external cycle while others remain unentrained. In fact, rats exposed to an artificially short 22 hr LD cycle express two stable circadian motor activity rhythms with different period lengths in individual animals. Our analysis of SCN gene expression under such conditions suggests that these two motor activity rhythms reflect the separate activities of two oscillators in the anatomically defined ventrolateral and dorsomedial SCN subdivisions. Our forced desychronization protocol has allowed the first stable separation of these two regional oscillators in vivo, correlating their activities to distinct behavioral outputs, and providing a powerful approach for understanding SCN tissue organization and signaling mechanisms in behaving animals.

Circadian desynchronization of core body temperature and sleep stages in the rat

Proper functioning of the human circadian timing system is crucial to physical and mental health. Much of what we know about this system is based on experimental protocols that induce the desynchronization of behavioral and physiological rhythms within individual subjects, but the neural (or extraneural) substrates for such desynchronization are unknown. We have developed an animal model of human internal desynchrony in which rats are exposed to artificially short (22-h) light–dark cycles. Under these conditions, locomotor activity, sleep–wake, and slow-wave sleep (SWS) exhibit two rhythms within individual animals, one entrained to the 22-h light–dark cycle and the other free-running with a period >24 h (τ>24 h). Whereas core body temperature showed two rhythms as well, further analysis indicates this variable oscillates more according to the τ>24 h rhythm than to the 22-h rhythm, and that this oscillation is due to an activity-independent circadian regulation. Paradoxical sleep (PS), on the other hand, shows only one free-running rhythm. Our results show that, similarly to humans, (i) circadian rhythms can be internally dissociated in a controlled and predictable manner in the rat and (ii) the circadian rhythms of sleep–wake and SWS can be desynchronized from the rhythms of PS and core body temperature within individual animals. This model now allows for a deeper understanding of the human timekeeping mechanism, for testing potential therapies for circadian dysrhythmias, and for studying the biology of PS and SWS states in a neurologically intact model.

Evolution of rat motor activity circadian rhythm under three different light patterns

The effects of the light pattern on the evolution of circadian motor activity rhythm of rats were studied in this work. Three different light patterns were used: LL (bright light, 300 lux), DD (dim red light) and LD (12:12 cycles). The animals used for the experiment were born and kept under each condition. At the day of weaning (21-22 days old) animals were isolated and their motor activity was detected by means of an inductive system. Data were recorded every 30 minutes for the first month after weaning. Periodogram analysis was applied to each animals data and the daily power spectra were calculated on the basis of the endogenous period, tau. The evolution of the rhythm was studied by examining the changes of the whole power spectra of the motor activity function, obtained by means of a Fourier analysis, through time. The power content of the circadian harmonic (PCCH) was considered to be a measure of the circadian character of the function. Results showed the predominancy of ultradian harmonics, when the animals were young, especially in LL, and the increase of the PCCH through time in all cases. Animals under LL show the circadian harmonic to be the main harmonic of the spectra at about day 15 after weaning, while the animals under DD show this harmonic as the main one from the first day. However, the power content of this harmonic increased until day 10. LD animals also showed the first harmonic as the main one from the time of weaning increasing the PCCH until day 7. These results are explained in respect to a multioscillatory system.(ABSTRACT TRUNCATED AT 250 WORDS)

Circadian Rhythms of Serum Concentrations of 12 Enzymes of Clinical Interest

A total of 25 apparently healthy adults (13 men and 12 women), 29.5 years (SD = 3.6 years) of age, served as subjects in a 24-h study conducted in Barcelona, Spain, in the spring of 1990. The group had a homogeneous pattern of meals, activity, and behavior. Six blood samples were collected at 4-h intervals over a single 24-h period beginning at 10:00 h. The oral temperature was measured at 2-h intervals to facilitate an independent biological time reference for the local population being studied. The serum concentration of 12 enzymes of clinical interest were measured in each sample: creatine kinase, creatine kinase 2, alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase, alkaline phosphatase, cholinesterase, lactate dehydrogenase, lactate dehydrogenase 1, 5-nucleotidase, pancreatic alpha-amylase, and triacylglycerol lipase. We supposed that all experimental data obtained for a quantity came from a single hypothetical subject that represented the central tendency of the population and then these data were analyzed for circadian rhythm by single cosinor. A statistically significant circadian rhythm was detected in all quantities studied (p < or = 0.05) except for serum concentrations of pancreatic alpha-amylase and triacylglycerol lipase. The maximum daily rhythmic variation was approximately 10% (interval, 6-14%) for all quantities studied except pancreatic alpha-amylase (2.6%). This rhythmic variation is greater than the analytical variation except for 5-nucleotidase and pancreatic alpha-amylase. The acrophases for the quantities studied (except that of triacylglycerol lipase) coincide with times near those of the oral temperature acrophase (18:01 local time). The results of this study will doubtless contribute to further documentation of the structure of the human circadian timing system and to establishment of time-qualified reference intervals for a defined group of subjects.

Noise-Induced Coherence in Multicellular Circadian Clocks

In higher organisms, circadian rhythms are generated by a multicellular genetic clock that is entrained very efficiently to the 24-h light-dark cycle. Most studies done so far of these circadian oscillators have considered a perfectly periodic driving by light, in the form of either a square wave or a sinusoidal modulation. However, in natural conditions, organisms are subject to nonnegligible fluctuations in the light level all through the daily cycle. In this article, we investigate how the interplay between light fluctuations and intercellular coupling affects the dynamics of the collective rhythm in a large ensemble of nonidentical, globally coupled cellular clocks modeled as Goodwin oscillators. On the basis of experimental considerations, we assume an inverse dependence of the cell-cell coupling strength on the light intensity, in such a way that the larger the light intensity, the weaker the coupling. Our results show a noise-induced rhythm generation for constant light intensities at which the clock is arrhythmic in the noise-free case. Importantly, the rhythm shows a resonancelike phenomenon as a function of the noise intensity. Such improved coherence can be only observed at the level of the overt rhythm and not at the level of the individual oscillators, thus suggesting a cooperative effect of noise, coupling, and the emerging synchronization between the oscillators.

Dissociation of the rat motor activity rhythm under T cycles shorter than 24 hours.

Since the suprachiasmatic nuclei (SCN) were identified as the principal mammalian circadian clock, many studies describing their morphology and physiology have been carried out. Today, the multioscillatory nature of the SCN, which explains the dissociation of the circadian rhythms under some experimental conditions, is widely accepted. Here, we study the simultaneous presence of two circadian rhythms in the motor activity of the rat when exposed to symmetric light-dark (LD) cycles shorter than 24 h (T21, T21.5, T22, T22.5, T23, and T23.5). One rhythmic component was entrained by the external LD cycle whereas the other ran free with a period longer than 24 h. The results show that two circadian rhythms were present only when T was shorter than T23, whereas at T23.5 only one entrained component was manifested. The manifestation of the two circadian components depends quantitatively on the period of the external cycle--i.e., the strength of the entrained rhythm increases when the external T is closer to 24 h--whereas that of the nonentrained rhythm decreases. The dissociation of the motor activity rhythm and the gradual appearance of the two components are explained by considering the entrainment of a multioscillatory system as not taking place as a whole but rather in a partial manner, in such a way that some oscillators may entrain but not others. The effect of the entrained oscillators is added to the masking effect of the LD cycles.

Entrainment of the rat motor activity rhythm: effects of the light–dark cycle and physical exercise

The circadian system is believed to be composed of a population of oscillators that couple together and generate a single rhythm. If this coupling is not strong enough, the circadian system can be dissociated into two or more groups of oscillators, and this is manifested in a dissociation of the overt rhythm into at least two circadian components. This study aims to examine the influence of factors, such as the difference in impact between T and tau, light intensity, and access to a running wheel, on the distribution of motor activity throughout the light-dark (LD) cycle and the dissociation of the rhythm. Rats were submitted to LD cycles of 23 h (T23) or 25 h. For each such cycle, half the rats were submitted to high light intensity and the other half to low light intensity. For each of these conditions, half the rats were kept in small cages, and the other half were in cages with a running wheel. Rats were maintained first under LD cycles and afterwards under constant darkness (DD). Motor activity was recorded throughout the whole experiment by means of activity meters with infrared beams. Results show that the distribution of motor activity throughout the cycle and the after effects observed in the rhythm under DD depended on light intensity and access to the wheel. Moreover, under T23, some rats showed two simultaneous circadian components whose manifestation also depended on the experimental conditions. The results indicate that the strength of circadian entrainment to LD cycles in the rat depends on three factors: the period length of the LD cycle, light intensity used during the light phase, and access to a running wheel.

Constant Bright Light (LL) during Lactation in Rats Prevents Arhythmicity Due to LL

Light has a strong effect on the circadian system. Light-dark (LD) cycles are the main zeitgebers for practically all organisms, and the exposure of animals to constant bright light (LL) alters the manifestation of circadian rhythms. In rats, exposure to LL in adulthood produces an arrhythmic pattern in their motor activity, with a large number of ultradian components. In previous experiments, we found that rats born and kept under LL during lactation develop, after weaning, a circadian rhythm which is maintained for at least a couple of months. Here, we examined motor activity rhythms under LL of two groups of rats which differed in the lighting conditions under which they were kept during lactation: 1) rats kept under LL during lactation (LL-rats), which manifested a circadian rhythm after weaning, and 2) rats kept under constant darkness (DD-rats), which were arrhythmic after weaning. We investigated whether the presence of rhythmicity under LL in LL-rats is a transitory effect or whether it persists throughout most of the life of the rat. Moreover, we examined motor activity rhythms of both groups of rats under different lighting conditions to find out other possible differences in the manifestation of their circadian rhythms. Results showed that there are no differences in the capacity of entrainment of both groups of rats to LD cycles or in the rhythm that rats show under DD. Most of the LL-rats maintained their circadian rhythms for the duration of the experiment (1 year), although we found differences in the rhythms manifested between males and females. We found that most of the LL-males became arrhythmic; consequently, at the end of the experiment, there were no differences in the number of males showing circadian rhythm in the LL- and DD-groups. Most of the females in the LL-group showed a clear circadian rhythm under LL during the entire experiment. Thus, LL during lactation has a protective effect against the disruptive effect of LL on the circadian rhythm, although it is only clearly manifested in females.

Light responses of the circadian system in leptin deficient mice.

Some evidences postulate a link between obesity and disturbances in circadian behavior. Here, we studied the manifestation of the circadian rhythm of motor activity and its response to light in the leptin deficiency model of obesity ob/ob mice. Motor activity in both ob/ob and wild type mice was first recorded in a small cage by activity meters with crossed infrared beams (IR) and later in a larger cage with a running wheel, where the number of wheel revolutions (WR) was also determined. Animals were maintained under light-dark (LD) or constant-dark (DD) conditions. We studied the free-running period and the rhythm profile, with special emphasis on the amount of activity in the dark and light phases of the LD cycle, and the phase and period responses to a light pulse. The results showed that ob/ob mice have a strong ultradian, rather than a circadian pattern, whose period range between 3 and 4.8h. Also, these animals showed a percentage of activity during light higher than controls. We did not find differences in the endogenous period of the circadian rhythm between mice groups in DD. However, ob/ob mice showed stronger phase delays after a light pulse at ZT15 than controls, and less masking effects in the transition from LD to DD compared with controls. This suggests a weaker circadian pacemaker of the ob/ob mice compared with controls.

Glucocorticoids Affect 24 h Clock Genes Expression in Human Adipose Tissue Explant Cultures

Aims to examine firstly whether CLOCK exhibits a circadian expression in human visceral (V) and subcutaneous (S) adipose tissue (AT) in vitro as compared with BMAL1 and PER2, and secondly to investigate the possible effect of the glucocorticoid analogue dexamethasone (DEX) on positive and negative clock genes expression. Subjects and Methods VAT and SAT biopsies were obtained from morbid obese women (body mass index≥40 kg/m2) (nu200a=u200a6). In order to investigate rhythmic expression pattern of clock genes and the effect of DEX on CLOCK, PER2 and BMAL1 expression, control AT (without DEX) and AT explants treated with DEX (2 hours) were cultured during 24 h and gene expression was analyzed at the following times: 10:00 h, 14:00 h, 18:00 h, 22:00 h, 02:00 h and 06:00 h, using qRT-PCR. Results CLOCK, BMAL1 and PER2 expression exhibited circadian patterns in both VAT and SAT explants that were adjusted to a typical 24 h sinusoidal curve. PER2 expression (negative element) was in antiphase with respect to CLOCK and in phase with BMAL1 expression (both positive elements) in the SAT (situation not present in VAT). A marked effect of DEX exposure on both positive and negative clock genes expression patterns was observed. Indeed, DEX treatment modified the rhythmicity pattern towards altered patterns with a period lower than 24 hours in all genes and in both tissues. Conclusions 24 h patterns in CLOCK and BMAL1 (positive clock elements) and PER2 (negative element) mRNA levels were observed in human adipose explants. These patterns were altered by dexamethasone exposure.