Nuclear receptor Rev-erb alpha (Nr1d1) functions in concert with Nr2e3 to regulate transcriptional networks in the retina.

The majority of diseases in the retina are caused by genetic mutations affecting the development and function of photoreceptor cells. The transcriptional networks directing these processes are regulated by genes such as nuclear hormone receptors. The nuclear hormone receptor gene Rev-erb alpha/Nr1d1 has been widely studied for its role in the circadian cycle and cell metabolism, however its role in the retina is unknown. In order to understand the role of Rev-erb alpha/Nr1d1 in the retina, we evaluated the effects of loss of Nr1d1 to the developing retina and its co-regulation with the photoreceptor-specific nuclear receptor gene Nr2e3 in the developing and mature retina. Knock-down of Nr1d1 expression in the developing retina results in pan-retinal spotting and reduced retinal function by electroretinogram. Our studies show that NR1D1 protein is co-expressed with NR2E3 in the outer neuroblastic layer of the developing mouse retina. In the adult retina, NR1D1 is expressed in the ganglion cell layer and is co-expressed with NR2E3 in the outer nuclear layer, within rods and cones. Several genes co-targeted by NR2E3 and NR1D1 were identified that include: Nr2c1, Recoverin, Rgr, Rarres2, Pde8a, and Nupr1. We examined the cyclic expression of Nr1d1 and Nr2e3 over a twenty-four hour period and observed that both nuclear receptors cycle in a similar manner. Taken together, these studies reveal a novel role for Nr1d1, in conjunction with its cofactor Nr2e3, in regulating transcriptional networks critical for photoreceptor development and function.

Efeito da luminosidade e do horário do exercício de natação até a exaustão na intensidade de lactato mínimo sobre respostas bioquímicas e hematológicas de ratos

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

The primate seahorse rhythm

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

Aspects of the activity rhythm and population size of troglophilic mygalomorph spiders (Trechona sp., Dipluridae) in a quartzite cave in Minas Gerais, Brazil

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Ecologia do movimento e dinâmica espaço-temporal da onça-pintada (Panthera onca) no Pantanal sul do Brasil

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

Influência da temperatura e do termoperíodo sobre a taxa metabólica de repouso em cascavéis, Crotalus durissus (Serpentes: Viperidae)

Snakes are ectothermic animals and, therefore, their physiological functions are strongly affected by temperature. For instance, the resting metabolic rate (RMR) of this animals increase with the rise in body temperature. However, metabolic determinations in ectothermic organisms, including snakes, are generally made by submitting the animals to constant temperature regimes. This experimental procedure, although widely used, accepted and certainly suitable in several cases, submit the animals to a very different situation from that experienced by them in nature. In fact, ectothermics are known by presenting extensive variations in their body temperatures trough the day and/or seasons. If this disagreement between the thermal biology of the animals and the experimental conditions, for instance over the circadian cycle, affects the determinations of metabolic rates of ectotherm animals, remains quite uncertain. Thus, this study aimed to test the effects of different thermal regimes (fluctuating vs constant) in different temperature ranges over the TMR of rattlesnakes (Crotalus durissus). Therefore, the TMR of rattlesnakes was measured by the oxygen consumption rates ( V O2) in the constant temperatures of 15°C, 20°C, 25°C, 30°C and 35°C. For fluctuating regimes, snakes were measured in thermoperiods of 12/12 hours, as follows: 15°C and 25°C; 20°C and 30°C; 25°C and 35°C. Our results show that the RMR of C. durissus rises as the temperature increases, regardless of the thermal regime. The obtained RMR in the constant regimes of 20°C and 25°C was not different from that measured in the correspondent fluctuating regimes (i.e., 15 - 25°C e 20 - 30°C). However, at constant 30°C, the RMR was significantly higher than that obtained in the 30°C fluctuating regime (25 - 35ºC). This indicates that the potential effects in submitting of snakes to different thermal regimes of its thermal biology become more important with...

Influência da temperatura e do termoperíodo sobre a taxa metabólica de repouso em cascavéis, Crotalus durissus (Serpentes: Viperidae)

Snakes are ectothermic animals and, therefore, their physiological functions are strongly affected by temperature. For instance, the resting metabolic rate (RMR) of this animals increase with the rise in body temperature. However, metabolic determinations in ectothermic organisms, including snakes, are generally made by submitting the animals to constant temperature regimes. This experimental procedure, although widely used, accepted and certainly suitable in several cases, submit the animals to a very different situation from that experienced by them in nature. In fact, ectothermics are known by presenting extensive variations in their body temperatures trough the day and/or seasons. If this disagreement between the thermal biology of the animals and the experimental conditions, for instance over the circadian cycle, affects the determinations of metabolic rates of ectotherm animals, remains quite uncertain. Thus, this study aimed to test the effects of different thermal regimes (fluctuating vs constant) in different temperature ranges over the TMR of rattlesnakes (Crotalus durissus). Therefore, the TMR of rattlesnakes was measured by the oxygen consumption rates ( V O2) in the constant temperatures of 15°C, 20°C, 25°C, 30°C and 35°C. For fluctuating regimes, snakes were measured in thermoperiods of 12/12 hours, as follows: 15°C and 25°C; 20°C and 30°C; 25°C and 35°C. Our results show that the RMR of C. durissus rises as the temperature increases, regardless of the thermal regime. The obtained RMR in the constant regimes of 20°C and 25°C was not different from that measured in the correspondent fluctuating regimes (i.e., 15 - 25°C e 20 - 30°C). However, at constant 30°C, the RMR was significantly higher than that obtained in the 30°C fluctuating regime (25 - 35ºC). This indicates that the potential effects in submitting of snakes to different thermal regimes of its thermal biology become more important with...

Daily modulation of the Heat shock proteins (Hsps) in three different species of scleractinian corals

Temperature and light intensity is the most important environmental parameters that influence circadian cycle of scleractinian corals. In this context, modulation of the biomarkers Hsp60 and Hsp70 in situ was investigated by three different healthy coral species (Acropora tenuis, Echinopora lamellosa and Porites lobata) not stress induced during time course of 24h. Significance species-specific modulation under natural conditions is displayed by all corals under study. A strong fluctuation in Hsps expression is shown by the most susceptible, branched coral A. tenuis, instead of fine and low modulation is shown by the massive coral P. lobata. From the results match between morphology difference and physiological difference response its suggest and similarity pattern between Hsps with different cellular compartments location is suggested too. Starting from this study health of coral reefs could be able to be investigated in the future with a set of biomarkers composed also by Hsps which will be set up.

The Effects of Prenatal Exposure to Altered Melatonin Levels on Hippocampal Gene Expression in the Male Rat

The stability of the circadian rhythm for mammals depends on the levels of serotonin and melatonin, neurohormones that signal for lightness and darkness, respectively. Disruption in the stability of neurohormones has been shown to be a critical factor in psychopathological disorders in humans. For example, altering levels of melatonin in utero through administration of melatonin or the melatonin receptor antagonist, luzindole, has been shown to cause changes in developmental growth and adult behavior in the male rat. Analysis of relative adult hippocampal gene expression with RT-PCR revealed differences in ARNTL expression that suggested abnormality in clock gene expression of the rats that were prenatally exposed to altered levels of melatonin. Differences in the degree of plasticity as suggested by previous behavior testing did not result in differences in gene expression for GABA receptors or NMDA receptors. Morevoer, growth associated protein 43, GAP-43, a protein that is necessary for neuronal growth cones as well as long term learning has been found to be critical for axon and presynaptic terminal formation and retention in other studies, but hippocampal gene expression in our study showed no significant alteration after exposure to various maternal melatonin levels. However, ARNTL is a key regulatory component of clock genes and the circadian cycle so that alterations in the expression of thi critical gene may lead to critical changes in neuronal growth and plasticity. Our data support the conclusion that the manipulation of maternal melatonin levels alters the brain development and the circadian cycles that may lead to physiological and behavioral abnormalities in adult offspring.

Altered emotional and locomotor responses in mice deficient in the transcription factor CREM

Various transcription factors act as nuclear effectors of the cAMP-dependent signaling pathway. These are the products of three genes in the mouse, CREB, CRE modulator (CREM), and ATF-1. CREM proteins are thought to play important roles within the hypothalamic–pituitary axis and in the control of rhythmic functions in the pineal gland. We have generated CREM-mutant mice and investigated their response in a variety of behavioral tests. CREM-null mice show a drastic increase in locomotion. In contrast to normal mice, the CREM-deficient mice show equal locomotor activity during the circadian cycle. The anatomy of the hypothalamic suprachiasmatic nuclei, the center of the endogenous pacemaker, is normal in mutant mice. Remarkably, CREM mutant mice also elicit a different emotional state, revealed by a lower anxiety in two different behavioral models, but they preserve the conditioned reactiveness to stress. These results demonstrate the high degree of functional specificity of each cAMP-responsive transcription factor in behavioral control.

Glucocorticoids have state-dependent stimulant effects on the mesencephalic dopaminergic transmission.

An increase in the activity of mesencephalic dopaminergic neurons has been implicated in the appearance of pathological behaviors such as psychosis and drug abuse. Several observations suggest that glucocorticoids might contribute to such an increase in dopaminergic activity. The present experiments therefore analyzed the effects of corticosterone, the major glucocorticoid in the rat, both on dopamine release in the nucleus accumbens of freely moving animals by means of microdialysis, and on locomotor activity, a behavior dependent on accumbens dopamine. Given that glucocorticoids have certain state-dependent neuronal effects, their action on dopamine was studied in situations differing in dopaminergic tonus, including during the light and dark phases of the circadian cycle, during eating, and in groups of animals differing in their locomotor reactivity to novelty. Dopaminergic activity is increased in the dark period, further increased during food-intake, and is higher in rats defined as high responders to novelty than in low responders. Corticosterone, peripherally administered in a dose that approximates stress-induced plasma concentrations, increased extracellular concentrations of dopamine, and this increase was augmented in the dark phase, during eating, and in high responder rats. Corticosterone had little or no effects in the light phase and in low responder rats. Corticosterone also stimulated locomotor activity, an effect that paralleled the release of dopamine and was abolished by neurochemical (6-hydroxydopamine) depletion of accumbens dopamine. In conclusion, glucocorticoids have state-dependent stimulant effects on mesencephalic dopaminergic transmission, and an interaction between these two factors might be involved in the appearance of behavioral disturbances.

Diel variation in movement patterns and habitat use by the near-threatened cabrera vole in mediterranean farmland

As variações circadianas no comportamento animal e o seu impacto nas populações constituem desafios importantes em ecologia e conservação. Nesta tese documentam-se as variações circadianas no uso do habitat e padrões de movimento pelo rato de Cabrera, em habitats Mediterrânicos fragmentados. O estudo baseou-se no radio-seguimento de indivíduos em habitats dominados por herbáceas e arbustos. Os resultados indicaram que a proporção de tempo despendido em deslocações, a distância percorrida, e a selecção do tipo de vegetação, estão fortemente interrelacionados, variando consideravelmente ao longo de diferentes períodos do dia. Os ratos movimentaram-se mais frequentemente e maiores distâncias nos períodos diurnos, durante os quais as áreas dominadas por herbáceas foram usadas mais intensivamente. Durante a estação seca houve alguma tendência para a diminuição dos movimentos durante as horas mais quentes. Estes resultados são discutidos no sentido de mostrar como indicadores comportamentais podem contribuir para melhorar a gestão e conservação da espécie; ABSTRACT: Understanding the circadian variations in species behaviour and its impacts on population is a challenging topic in ecology and conservation. This thesis documents the circadian variations in habitat use and movement patterns by Cabrera voles in fragmented Mediterranean farmland. The study was based on radiotracking data of individuals living in habitat patches dominated by wet grasses and shrubs. Results indicated that the proportion of time animals spent moving, the distance moved and the selection strength of vegetation were closely linked behavioural traits, which varied considerably across the 24 hour cycle. Voles moved more frequently and over larger distances during daytime, which was when wet grasses were used more intensively. During the dry season there was some tendency for a decrease in movement activity during the hottest hours of the day. These results are used to discuss how behavioural indicators may be useful to improve conservation management of the species.

Intrinsically photosensitive melanopsin retinal ganglion cell contributions to the post-illumination pupil response and circadian rhythm

Intrinsically photosensitive retinal ganglion cells (ipRGCs) in the eye transmit the environmental light level, projecting to the suprachiasmatic nucleus (SCN) (Berson, Dunn & Takao, 2002; Hattar, Liao, Takao, Berson & Yau, 2002), the location of the circadian biological clock, and the olivary pretectal nucleus (OPN) of the pretectum, the start of the pupil reflex pathway (Hattar, Liao, Takao, Berson & Yau, 2002; Dacey, Liao, Peterson, Robinson, Smith, Pokorny, Yau & Gamlin, 2005). The SCN synchronizes the circadian rhythm, a cycle of biological processes coordinated to the solar day, and drives the sleep/wake cycle by controlling the release of melatonin from the pineal gland (Claustrat, Brun & Chazot, 2005). Encoded photic input from ipRGCs to the OPN also contributes to the pupil light reflex (PLR), the constriction and recovery of the pupil in response to light. IpRGCs control the post-illumination component of the PLR, the partial pupil constriction maintained for > 30 sec after a stimulus offset (Gamlin, McDougal, Pokorny, Smith, Yau & Dacey, 2007; Kankipati, Girkin & Gamlin, 2010; Markwell, Feigl & Zele, 2010). It is unknown if intrinsic ipRGC and cone-mediated inputs to ipRGCs show circadian variation in their photon-counting activity under constant illumination. If ipRGCs demonstrate circadian variation of the pupil response under constant illumination in vivo, when in vitro ipRGC activity does not (Weng, Wong & Berson, 2009), this would support central control of the ipRGC circadian activity. A preliminary experiment was conducted to determine the spectral sensitivity of the ipRGC post-illumination pupil response under the experimental conditions, confirming the successful isolation of the ipRGC response (Gamlin, et al., 2007) for the circadian experiment. In this main experiment, we demonstrate that ipRGC photon-counting activity has a circadian rhythm under constant experimental conditions, while direct rod and cone contributions to the PLR do not. Intrinsic ipRGC contributions to the post-illumination pupil response decreased 2:46 h prior to melatonin onset for our group model, with the peak ipRGC attenuation occurring 1:25 h after melatonin onset. Our results suggest a centrally controlled evening decrease in ipRGC activity, independent of environmental light, which is temporally synchronized (demonstrates a temporal phase-advanced relationship) to the SCN mediated release of melatonin. In the future the ipRGC post-illumination pupil response could be developed as a fast, non-invasive measure of circadian rhythm. This study establishes a basis for future investigation of cortical feedback mechanisms that modulate ipRGC activity.

Circadian Rhythm Disruptions and Health

The doctoral thesis defined connections between circadian rhythm disruptions and health problems. Sleep debt, jet-lag, shift work, as well as transitions into and out of the daylight saving time may lead to circadian rhythm disruptions. Disturbed circadian rhythm causes sleep deprivation and decrease of mood and these effects may lead to higher accident rates and trigger mental illnesses. Circadian clock genes are involved in the regulation of the cell cycle and metabolism and thus unstable circadian rhythmicity may also lead to cancer development. In publications I-III it was explored how transitions into and out of the daylight saving time impact the sleep efficiency and the rest-activity cycles of healthy individuals. Also it was explored whether the effect of transition is different in fall as compared to spring, and whether there are subgroup specific differences in the adjustment to transitions into and out of daylight saving time. The healthy participants of studies I-III used actigraphs before and after the transitions and filled in the morningness-eveningness and seasonal pattern assessment questionnaires. In publication IV the incidence of hospital-treated accidents and manic episodes was explored two weeks before and two weeks after the transitions into and out of the daylight saving time in years 1987-2003. In publication V the relationship between circadian rhythm disruption and the prevalence of Non-Hodgkin lymphoma was studied. The study V consisted of all working aged Finns who participated in the national population census in 1970. For our study, all the cancers diagnosed during the years 1971-1995 were extracted from the Finnish Cancer Register and linked with the 1970 census files. In studies I-III it was noticed that transitions into and out of the daylight saving time disturbs the sleep-wake cycle and the sleep efficiency of the healthy participants. We also noticed that short sleepers were more sensitive than long sleepers for sudden changes in the circadian rhythm. Our results also indicated that adaptation to changes in the circadian rhythm is potentially sex, age and chronotype-specific. In study IV no significant increase in the occurrence of hospital treated accidents or manic episodes was noticed. However, interesting observations about the seasonal fluctuation of the occurrence rates of accidents and manic episodes were made. Study V revealed that there might be close relationship between circadian rhythm disruption and cancer. The prevalence of Non-Hodgkin lymphoma was the highest among night workers. The five publications included in this thesis together point out that disturbed circadian rhythms may have adverse effect on health. Disturbed circadian rhythms decrease the quality of sleep and weaken the sleep-wake cycle. A continuous circadian rhythm disruption may also predispose individuals to cancer development. Since circadian rhythm disruptions are common in modern society they might have a remarkable impact on the public health. Thus it is important to continue circadian rhythm research so that better prevention and treatment methods can be developed. Keywords: Circadian rhythm, daylight saving time, manic episodes, accidents, Non-Hodgkin lymphoma 11

The circadian oscillator gene GIGANTEA mediates a long-term response of the Arabidopsis thaliana circadian clock to sucrose.

Circadian clocks are 24-h timing devices that phase cellular responses; coordinate growth, physiology, and metabolism; and anticipate the day-night cycle. Here we report sensitivity of the Arabidopsis thaliana circadian oscillator to sucrose, providing evidence that plant metabolism can regulate circadian function. We found that the Arabidopsis circadian system is particularly sensitive to sucrose in the dark. These data suggest that there is a feedback between the molecular components that comprise the circadian oscillator and plant metabolism, with the circadian clock both regulating and being regulated by metabolism. We used also simulations within a three-loop mathematical model of the Arabidopsis circadian oscillator to identify components of the circadian clock sensitive to sucrose. The mathematical studies identified GIGANTEA (GI) as being associated with sucrose sensing. Experimental validation of this prediction demonstrated that GI is required for the full response of the circadian clock to sucrose. We demonstrate that GI acts as part of the sucrose-signaling network and propose this role permits metabolic input into circadian timing in Arabidopsis.