Jacopo Aguzzi

Evidence for an Overlapping Role of CLOCK and NPAS2 Transcription Factors in Liver Circadian Oscillators

ABSTRACT The mechanisms underlying the circadian control of gene expression in peripheral tissues and influencing many biological pathways are poorly defined. Factor VII (FVII), the protease triggering blood coagulation, represents a valuable model to address this issue in liver since its plasma levels oscillate in a circadian manner and its promoter contains E-boxes, which are putative DNA-binding sites for CLOCK-BMAL1 and NPAS2-BMAL1 heterodimers and hallmarks of circadian regulation. The peaks of FVII mRNA levels in livers of wild-type mice preceded those in plasma, indicating a transcriptional regulation, and were abolished in Clock−/−; Npas2−/− mice, thus demonstrating a role for CLOCK and NPAS2 circadian transcription factors. The investigation of Npas2−/− and ClockΔ19/Δ19 mice, which express functionally defective heterodimers, revealed robust rhythms of FVII expression in both animal models, suggesting a redundant role for NPAS2 and CLOCK. The molecular bases of these observations were established through reporter gene assays. FVII transactivation activities of the NPAS2-BMAL1 and CLOCK-BMAL1 heterodimers were (i) comparable (a fourfold increase), (ii) dampened by the negative circadian regulators PER2 and CRY1, and (iii) abolished upon E-box mutagenesis. Our data provide the first evidence in peripheral oscillators for an overlapping role of CLOCK and NPAS2 in the regulation of circadianly controlled genes.

Spontaneous internal desynchronization of locomotor activity and body temperature rhythms from plasma melatonin rhythm in rats exposed to constant dim light

Background We have recently reported that spontaneous internal desynchronization between the locomotor activity rhythm and the melatonin rhythm may occur in rats (30% of tested animals) when they are maintained in constant dim red light (LLdim) for 60 days. Previous work has also shown that melatonin plays an important role in the modulation of the circadian rhythms of running wheel activity (Rw) and body temperature (Tb). The aim of the present study was to investigate the effect that desynchronization of the melatonin rhythm may have on the coupling and expression of circadian rhythms in Rw and Tb. Methods Rats were maintained in a temperature controlled (23–24°C) ventilated lightproof room under LLdim (red dim light 1 μW/cm2 [5 Lux], lower wavelength cutoff at 640 nm). Animals were individually housed in cages equipped with a running wheel and a magnetic sensor system to detect wheel rotation; Tb was monitored by telemetry. Tb and Rw data were recorded in 5-min bins and saved on disk. For each animal, we determined the mesor and the amplitude of the Rw and Tb rhythm using waveform analysis on 7-day segments of the data. After sixty days of LLdim exposure, blood samples (80–100 μM) were collected every 4 hours over a 24-hrs period from the tail artery, and serum melatonin levels were measured by radioimmunoassay. Results Twenty-one animals showed clear circadian rhythms Rw and Tb, whereas one animal was arrhythmic. Rw and Tb rhythms were always strictly associated and we did not observe desynchronization between these two rhythms. Plasma melatonin levels showed marked variations among individuals in the peak levels and in the night-to-day ratio. In six rats, the night-to-day ratio was less than 2, whereas in the rat that showed arrhythmicity in Rw and Tb melatonin levels were high and rhythmic with a large night-to-day ratio. In seven animals, serum melatonin levels peaked during the subjective day (from CT0 to CT8), thus suggesting that in these animals the circadian rhythm of serum melatonin desynchronized from the circadian rhythms of Rw and Tb. No significant correlation was observed between the amplitude (or the levels) of the melatonin profile and the amplitude and mesor of the Rw and Tb rhythms. Conclusion Our data indicate that the free-running periods (τ) and the amplitude of Rw and Tb were not different between desynchronized and non-desynchronized rats, thus suggesting that the circadian rhythm of serum melatonin plays a marginal role in the regulation of the Rw and Tb rhythms. The present study also supports the notion that in the rat the circadian rhythms of locomotor activity and body temperature are controlled by a single circadian pacemaker.

LOCOMOTOR ACTIVITY RHYTHMS OF CONTINENTAL SLOPE NEPHROPS NORVEGICUS (DECAPODA: NEPHROPIDAE)

Abstract The locomotor rhythmicity of the Norway lobster Nephrops norvegicus was studied under constant conditions of darkness in individuals collected on the continental slope (400–430 m). Periodogram analysis revealed the occurrence of both circadian (of around 24 h) and ultradian (of around 12 and 18 h) periodicities. Form estimate analysis of the circadian and ultradian time series revealed the occurrence of significant peaks of activity during the expected night phase of the cycle and day-night transitions, respectively. No ultradian locomotor activity rhythms have been reported in previous studies on continental shelf N. norvegicus, suggesting that this phenomenon may be limited to deep-water animals. A discussion is presented to account for the occurrence of the mechanism of ultradian rhythms when there is significant environmental light intensity reduction, as on the continental slope, where the species attains its maximum densities in the western Mediterranean.

Effect of Long-Term Exposure to Constant Dim Light on the Circadian System of Rats

The newly discovered multi-oscillatory nature of the mammalian circadian clock system and the cloning of the genes involved in the molecular mechanism that generates circadian rhythmicity have opened new approaches for understanding how mammals are temporally organized and how the mammalian circadian system reacts to the lack of normal synchronization cues. In the present study we investigated the effects of long-term exposure to constant red dim light on the pattern of the expression of Period 1 in the suprachiasmatic nuclei of the hypothalamus and of Arylalkylamine N-acetyltransferase(Aa-nat) in the retina and pineal gland. Our data demonstrate that Period 1 mRNA expression in the suprachiasmatic nuclei of the hypothalamus was not affected by exposure to constant red dim light for 60 days, whereas Aa-nat mRNA expression in the retina and in the pineal gland was significantly affected, since in some animals (20–30%) Aa-nat mRNA levels were found to be higher during the subjective day. A circadian rhythm of serum melatonin and locomotor activity was present in all the animals tested. In 4 animals serum melatonin levels were high during the subjective day. Our data suggest that long-term exposure to constant red dim light may induce desynchronization between the circadian rhythm of locomotor activity and serum melatonin levels.

Effect of photoreceptor degeneration on circadian photoreception and free-running period in the Royal College of Surgeons rat

The study of how the retina processes the photic information required for the entrainment of the circadian system is an exciting new topic in retinal neurobiology. We have recently shown that in RCS/N-rdy rats melanopsin mRNA levels are dramatically reduced (about 90%) and melanopsin immunoreactivity cannot be detected in the retina of these rats at 60 days of age. Although RCS/N-rdy rats are a widely used model to investigate mechanisms of photoreceptor degeneration, no study has investigated circadian photoreception in these animals. The aim of this study was to examine circadian photoreception in RCS/N-rdy(+) (rdy(+)) rats homozygous for the normal rdy allele and age-matched RCS/N-rdy (rdy) homozygotes with retinal dystrophy. No differences between RCS/N-rdy and rdy(+) were observed in light-induced phase shift of locomotor activity at the three light intensities used (1 x 10(-3), 1 x 10(-1), and 1 x 10(1) microW cm(-2)). Surprisingly, we observed that in RCS/N-rdy the free-running period of the circadian rhythm of locomotor activity was shorter (P<0.01) than in rdy(+), thus suggesting that photoreceptor degeneration may affect the free-running period of the locomotor activity rhythm.

Cardiac Activity of Nephrops Norvegicus (Decapoda: Nephropidae): The Relationship between Circadian and Ultradian Rhythms

Abstract The relationship between rhythms of circadian periodicity (i.e., from 20 to 28 h) and ultradian periodicity (i.e., less than 20 h) was studied in the burrowing decapod Nephrops norvegicus at the level of its cardiac activity. Animals were kept over a month under constant darkness (DD) interrupted by a few days of light-darkness regime (LD) at the beginning of the experiment. Time series (beats per 10 min) were subdivided into stages of similar numbers of days. A general mean waveform was computed per stage by averaging 24 h segments of different time series per corresponding 10 min intervals. Marked fluctuations were observed at the beginning of tests in DD, being disrupted during animals exposure to LD. Fluctuations progressively recovered over following stages of DD. The activity part of a rhythm (α) was computed in mean waveforms of different time series per each stage. Resulting values were averaged at corresponding stages. A significant increment of mean α was observed from DD to LD, decreasing over the following prolonged DD exposure. Periodogram analysis was used to assess periodicities of time series at each stage. Fourier analysis was undertaken to assess the transformation of cardiac rhythms over consecutive stages not only in terms of periodicity but also as amplitude. Both analyses showed the presence of different circadian and ultradian (i.e., 12 h and 18 h) rhythms varying in their amplitude at different stages of testing. A preponderance of time series with ultradian periodicities took place in the first stage of DD. Under LD, the number of time series showing 18 h periodicity increased, but their amplitude of fluctuation was lower compared to the previous stage. In contrast, the circadian periodicity present in the first stage of DD disappeared in LD, to be restored over prolonged DD exposure. Present results suggest that a disruption of the circadian rhythm in cardiac activity generated ultradian periods when controlling oscillators became uncoupled. Results are discussed in the context of the ecology of the species, and a model based on the phase decoupling of circadian oscillators is presented to account for the generation of ultradian 12 h and 18 h periodicities.

Effect of space flight on circadian rhythms.

Neuroscience Institute, Morehouse School of Medicine, Atlanta, GA, USAIntroductionDaily rhythmicity is a ubiquitous feature of living systems. Generally, theserhythms are not just passive consequences of cyclic fluctuations in the environ-ment, but, rather they originate within the organisms. They are generatedand controlled by endogenous physiological oscillators. The fundamentaladaptive function of an endogenously programmed rhythmicity is to providean optimal and anticipatory temporal organization of physiological processesand behavior in relation to the environment. Endogenously controlled dailyrhythms are called circadian rhythms, referring to the fact that they have aperiod of about a day. Under conditions of temporal isolation this period,in most cases, slightly deviates from exactly 24 h. Synchronization of endo-genous circadian rhythms to the environment occurs through daily adjustmentof the clock by external time cues or zeitgebers. The most important and reliablecue for synchronization is the daily light–dark cycle (Pittendrigh and Daan,1976).Properties of circadian rhythmsSince some of the terminology used in this review is specific to the circadianspecialty, we will begin this chapter with an explanation of the most commonterms used to describe circadian rhythms. A circadian clock is a part of anorganism which is capable of generating a self-sustained rhythm with a periodclose to 24 h. A rhythm is characterized by three features: period, amplitudeand phase (Fig. 1). The period is the length of time necessary to completeone full cycle, the amplitude is, roughly, the difference between the maximumand the minimum levels observed during a full cycle, and the phase refers to thetemporal relationship between a specific identifiable point on the cycle and apoint on a reference cycle.When an organism is maintained under constant conditions its circadianrhythms persist with a period close to, but different from 24 h. The fact thatrhythmicity persists under constant condition demonstrates that the rhythmis endogenous. The period of a rhythm under constant conditions is called its

The next expansion of NEMO-SN1, EMSO node, by video imaging equipment for the monitoring of the local deep-sea communities

Aguzzi, Jacopo... et. al.-- European Multidisciplinary Seafloor Observatory Conference Ocean Observatories Challenges and Progress (EMSO Conference OOCP), Scientific ideas, early results and infrastructure development, 13-15 November 2013, Rome.-- 1 page