Serotonin and circadian rhythms

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

CIRCADIAN OSCILLATORY PATTERNS OF OXYGEN-UPTAKE IN INDIVIDUAL WORKERS OF THE ANT CAMPONOTUS-RUFIPES

Respiratory rates of individual workers of Camponotus rufipes Fabricius (Hymenoptera: Formicidae) were measured at 25-degrees-C and LD 12:12 h (lights on 06.00 hours), DL 12:12 h (lights on 18.00 hours), LL (850 lux) and DD (red light, 20-30 lux), using the micro-Warburg technique. Worker ants were collected from natural nest during the winter of 1987 in a woodland park in the region of Rio Claro, São Paulo, Brazil. The respiration of ants showed a circadian rhythm with acrophase ranging from 20 h 41 min to 01 h 18 min and from 10 h 32 min to 12 h 22 min at LD and DD, respectively. In constant darkness the rhythmometric variables were similar to those presented by ants kept at LD 12:12 h. Under constant light no circadian rhythm in the respiration rates was found. A reduction in the amplitude was observed, indicating an inhibitory effect of this light regime on the respiration process.

Circadian time-dependent effects of fencamfamine on inhibition of dopamine uptake and release in rat striatal slices

Fencamfamine (FCF) is a central stimulant that facilitates central dopaminergic transmission through inhibition of dopamine uptake and enhanced release of the transmitter. We evaluated the changes in the inhibition of uptake and the release of striatal [ 3H]-dopamine at 9:00 and 21:00 h, times corresponding to maximal and minimal behavioral responses to FCF, respectively. Adult male Wistar rats (200-250 g) maintained on a 12-h light/12-h dark cycle (lights on at 7:00 h) were used. In the behavioral experiments the rats (N = 8 for each group) received FCF (3.5 mg/kg, ip) or saline at 9:00 or 21:00 h. Fifteen minutes after treatment the duration of activity (sniffing, rearing and locomotion) was recorded for 120 min. The basal motor activity was higher (28.6 ± 4.2 vs 8.4 ± 3.5 s) after saline administration at 21:00 h than at 9:00 h. FCF at a single dose significantly enhanced the basal motor activity (38.3 ± 4.5 vs 8.4 ± 3.5 s) and increased the duration of exploratory activity (38.3 ± 4.5 vs 32.1 ± 4.6 s) during the light, but not the dark phase. Two other groups of rats (N = 6 for each group) were decapitated at 9:00 and 21:00 h and striata were dissected for dopamine uptake and relase assays. The inhibition of uptake and release of [ 3H]-dopamine were higher at 9:00 than at 21:00 h, suggesting that uptake inhibition and the release properties of FCF undergo daily variation. These data suggest that the circadian time-dependent effects of FCF might be related to a higher susceptibility of dopamine presynaptic terminals to the action of FCF during the light phase which corresponds to the rats' resting period.

Do Circadian Preferences Influence the Sleep Patterns of Night Shift Drivers?

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Evolutionary History of the PER3 Variable Number of Tandem Repeats (VNTR): Idiosyncratic Aspect of Primate Molecular Circadian Clock

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Free-running circadian rhythms of muscle strength, reaction time, and body temperature in totally blind people

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Circadian and seasonal variation of the body temperature of sheep in a tropical environment.

Nychthemeral and annual rhythms of the rectal temperature were determined for Corriedale sheep in a tropical climate. The minimum rectal temperature averaged 39.55 degrees C at 0500 hours in summer, and 38.87 degrees C at 0600 hours in winter. The maximum was 40.03 degrees C in summer (1700 hours) and 39.33 degrees C in winter (1830 hours). Annual cycle of the rectal temperature showed a minimum in July and maximum in December.

Respiratory rhythms in stingless bee workers: circadian and ultradian components throughout adult development

The workers of the stingless bee, Melipona quadrifasciata, assume different tasks during their adult life. Newly emerged individuals remain inside the nest, without contact with the external environment. Maturing workers go to more peripheral regions and only the oldest, the foragers, leave the nest. As this diversity of activities implies different metabolic patterns, oxygen consumption has been measured in workers of three different ages: 24-48 h (nurses), 10-15 days (builders), and older than 25 days (foragers). Oxygen consumption of individually isolated workers was determined by intermittent respirometry, under constant darkness and temperature of 25 +/- 1 degrees C. Sets of 24-h measurements were obtained from individuals belonging to each of the three worker groups. Rhythmicity has been assessed in the daily (24 h) and ultradian (5-14 h) domains. This experimental design allowed detection of endogenous rhythms without the influence of the social group and without inflicting stress on the individuals, as would be caused by their longer isolation from the colony. Significant 24-h rhythms in oxygen consumption were present in nurses, builders and foragers; therefore, workers are rhythmic from the age of 24-48 h. However, the amplitude of the circadian rhythm changed according to age: nurses showed the lowest values, while foragers consistently presented the largest ones, about ten times larger than the amplitude of nurses` respiratory rhythm. Ultradian frequencies were detected for all worker groups, the power and frequencies of which varied little with age. This means that the ultradian strength was relatively larger in nurses and apparently maintains some relationship with the queen`s oviposition episodes.

Melanopsin Retinal Ganglion Cells: relevance to circadian rhythms and sleep in neurodegeneration

In this PhD thesis 3 projects were addressed focusing on the melanopsin retinal ganglion cells (mRGCs) system and its relevance for circadian rhythms and sleep in neurodegeneration. The first project was aimed at completing the characterization of mRGCs system in hereditary optic neuropathies (LHON and DOA). We confirmed that mRGCs are relatively spared also in post-mortem retinal specimens of a DOA case and pupillometric evaluation of LHON patients showed preservation of the pupillary light reflex, with attenuated responses compared to controls. Cell studies failed to indicate a protective role exerted by melanopsin itself. The second project was aimed at characterizing the possible occurrence of optic neuropathy and rest-activity circadian rhythm dysfunction in Alzheimer (AD) and Parkinson disease (PD), as well as, at histological level, the possible involvement of mRGCs in AD. OCT studies demonstrated a subclinical optic neuropathy in both AD and PD patients, with a different pattern involving the superior and nasal quadrants in AD and the temporal quadrant in PD. Actigraphic studies demonstrated a tendency towards an increased intradaily variability (IV) and reduced relative amplitude (RA) of rest-activity circadian rhythm in AD and a significant increased IV a reduced RA in PD. Immunohistochemical analysis of post-mortem retinal specimens and optic nerve cross-sections of neuropathologically confirmed AD cases demonstrated a significant loss of mRGCs and a nearly significant loss of axons in AD compared to controls. The mRGCs were affected in AD independently from age and magnitude of axonal loss. Overall these results suggest a role of the mRGCs system in the pathogenesis of circadian dysfunction in AD. The third project was aimed at evaluating the possible association between a single nucleotide polymorphism of the OPN4 gene and chronotype or SAD, failing to find any significant association with chronotype, but showing a non-significant increment of TT genotype in SAD.

Simulation of Drosophila circadian oscillations, mutations, and light responses by a model with VRI, PDP-1, and CLK.

A model of Drosophila circadian rhythm generation was developed to represent feedback loops based on transcriptional regulation of per, Clk (dclock), Pdp-1, and vri (vrille). The model postulates that histone acetylation kinetics make transcriptional activation a nonlinear function of [CLK]. Such a nonlinearity is essential to simulate robust circadian oscillations of transcription in our model and in previous models. Simulations suggest that two positive feedback loops involving Clk are not essential for oscillations, because oscillations of [PER] were preserved when Clk, vri, or Pdp-1 expression was fixed. However, eliminating positive feedback by fixing vri expression altered the oscillation period. Eliminating the negative feedback loop in which PER represses per expression abolished oscillations. Simulations of per or Clk null mutations, of per overexpression, and of vri, Clk, or Pdp-1 heterozygous null mutations altered model behavior in ways similar to experimental data. The model simulated a photic phase-response curve resembling experimental curves, and oscillations entrained to simulated light-dark cycles. Temperature compensation of oscillation period could be simulated if temperature elevation slowed PER nuclear entry or PER phosphorylation. The model makes experimental predictions, some of which could be tested in transgenic Drosophila.

A reduced model clarifies the role of feedback loops and time delays in the Drosophila circadian oscillator.

Although several detailed models of molecular processes essential for circadian oscillations have been developed, their complexity makes intuitive understanding of the oscillation mechanism difficult. The goal of the present study was to reduce a previously developed, detailed model to a minimal representation of the transcriptional regulation essential for circadian rhythmicity in Drosophila. The reduced model contains only two differential equations, each with time delays. A negative feedback loop is included, in which PER protein represses per transcription by binding the dCLOCK transcription factor. A positive feedback loop is also included, in which dCLOCK indirectly enhances its own formation. The model simulated circadian oscillations, light entrainment, and a phase-response curve with qualitative similarities to experiment. Time delays were found to be essential for simulation of circadian oscillations with this model. To examine the robustness of the simplified model to fluctuations in molecule numbers, a stochastic variant was constructed. Robust circadian oscillations and entrainment to light pulses were simulated with fewer than 80 molecules of each gene product present on average. Circadian oscillations persisted when the positive feedback loop was removed. Moreover, elimination of positive feedback did not decrease the robustness of oscillations to stochastic fluctuations or to variations in parameter values. Such reduced models can aid understanding of the oscillation mechanisms in Drosophila and in other organisms in which feedback regulation of transcription may play an important role.

Mechanism-based inhibition of the melatonin rhythm enzyme: Pharmacologic exploitation of active site functional plasticity

Serotonin N-acetyltransferase is the enzyme responsible for the diurnal rhythm of melatonin production in the pineal gland of animals and humans. Inhibitors of this enzyme active in cell culture have not been reported previously. The compound N-bromoacetyltryptamine was shown to be a potent inhibitor of this enzyme in vitro and in a pineal cell culture assay (IC50 ≈ 500 nM). The mechanism of inhibition is suggested to involve a serotonin N-acetyltransferase-catalyzed alkylation reaction between N-bromoacetyltryptamine and reduced CoA, resulting in the production of a tight-binding bisubstrate analog inhibitor. This alkyltransferase activity is apparently catalyzed at a functionally distinct site compared with the acetyltransferase activity active site on serotonin N-acetyltransferase. Such active site plasticity is suggested to result from a subtle conformational alteration in the protein. This plasticity allows for an unusual form of mechanism-based inhibition with multiple turnovers, resulting in “molecular fratricide.” N-bromoacetyltryptamine should serve as a useful tool for dissecting the role of melatonin in circadian rhythm as well as a potential lead compound for therapeutic use in mood and sleep disorders.

Pituitary adenylyl cyclase-activating peptide: A pivotal modulator of glutamatergic regulation of the suprachiasmatic circadian clock

The circadian clock in the suprachiasmatic nucleus (SCN) of the hypothalamus organizes behavioral rhythms, such as the sleep–wake cycle, on a near 24-h time base and synchronizes them to environmental day and night. Light information is transmitted to the SCN by direct retinal projections via the retinohypothalamic tract (RHT). Both glutamate (Glu) and pituitary adenylyl cyclase-activating peptide (PACAP) are localized within the RHT. Whereas Glu is an established mediator of light entrainment, the role of PACAP is unknown. To understand the functional significance of this colocalization, we assessed the effects of nocturnal Glu and PACAP on phasing of the circadian rhythm of neuronal firing in slices of rat SCN. When coadministered, PACAP blocked the phase advance normally induced by Glu during late night. Surprisingly, blocking PACAP neurotransmission, with either PACAP6–38, a specific PACAP receptor antagonist, or anti-PACAP antibodies, augmented the Glu-induced phase advance. Blocking PACAP in vivo also potentiated the light-induced phase advance of the rhythm of hamster wheel-running activity. Conversely, PACAP enhanced the Glu-induced delay in the early night, whereas PACAP6–38 inhibited it. These results reveal that PACAP is a significant component of the Glu-mediated light-entrainment pathway. When Glu activates the system, PACAP receptor-mediated processes can provide gain control that generates graded phase shifts. The relative strengths of the Glu and PACAP signals together may encode the amplitude of adaptive circadian behavioral responses to the natural range of intensities of nocturnal light.

Identification of a novel vertebrate circadian clock-regulated gene encoding the protein nocturnin

Photoreceptors of the Xenopus laevis retina are the site of a circadian clock. As part of a differential display screen for rhythmic gene products in this system, we have identified a photoreceptor-specific mRNA expressed in peak abundance at night. cDNA cloning revealed an open reading frame encoding a putative 388 amino acid protein that we have named “nocturnin” (for night-factor). This protein has strong sequence similarity to the C-terminal domain of the yeast transcription factor, CCR4, as well as a leucine zipper-like dimerization motif. Nocturnin mRNA levels exhibit a high amplitude circadian rhythm and nuclear run-on analysis indicates that it is controlled by the retinal circadian clock at the level of transcription. Our observations suggest that nocturnin may function through protein–protein interaction either as a component of the circadian clock or as a downstream effector of clock function.

Integration of circadian and phototransduction pathways in the network controlling CAB gene transcription in Arabidopsis

The transcription of CAB genes, encoding the chlorophyll a/b-binding proteins, is rapidly induced in dark-grown Arabidopsis seedlings following a light pulse. The transient induction is followed by several cycles of a circadian rhythm. Seedlings transferred to continuous light are known to exhibit a robust circadian rhythm of CAB expression. The precise waveform of CAB expression in light–dark cycles, however, reflects a regulatory network that integrates information from photoreceptors, from the circadian clock and possibly from a developmental program. We have used the luciferase reporter system to investigate CAB expression with high time resolution. We demonstrate that CAB expression in light-grown plants exhibits a transient induction following light onset, similar to the response in dark-grown seedlings. The circadian rhythm modulates the magnitude and the kinetics of the response to light, such that the CAB promoter is not light responsive during the subjective night. A signaling pathway from the circadian oscillator must therefore antagonize the phototransduction pathways controlling the CAB promoter. We have further demonstrated that the phase of maximal CAB expression is delayed in light–dark cycles with long photoperiods, due to the entrainment of the circadian oscillator. Under short photoperiods, this pattern of entrainment ensures that dawn coincides with a phase of high light responsiveness, whereas under long photoperiods, the light response at dawn is reduced.