Women temporarily synchronize their menstrual cycles with the luminance and gravimetric cycles of the Moon.

Many species synchronize reproductive behavior with a particular phase of the lunar cycle to increase reproductive success. In humans, a lunar influence on reproductive behavior remains controversial, although the human menstrual cycle has a period close to that of the lunar cycle. Here, we analyzed long-term menstrual recordings of individual women with distinct methods for biological rhythm analysis. We show that women's menstrual cycles with a period longer than 27 days were intermittently synchronous with the Moon's luminance and/or gravimetric cycles. With age and upon exposure to artificial nocturnal light, menstrual cycles shortened and lost this synchrony. We hypothesize that in ancient times, human reproductive behavior was synchronous with the Moon but that our modern lifestyles have changed reproductive physiology and behavior.

Neuronal circadian clock protein oscillations are similar in behaviourally rhythmic forager honeybees and in arrhythmic nurses.

Internal clocks driving rhythms of about a day (circadian) are ubiquitous in animals, allowing them to anticipate environmental changes. Genetic or environmental disturbances to circadian clocks or the rhythms they produce are commonly associated with illness, compromised performance or reduced survival. Nevertheless, some animals including Arctic mammals, open sea fish and social insects such as honeybees are active around-the-clock with no apparent ill effects. The mechanisms allowing this remarkable natural plasticity are unknown. We generated and validated a new and specific antibody against the clock protein PERIOD of the honeybee Apis mellifera (amPER) and used it to characterize the circadian network in the honeybee brain. We found many similarities to Drosophila melanogaster and other insects, suggesting common anatomical organization principles in the insect clock that have not been appreciated before. Time course analyses revealed strong daily oscillations in amPER levels in foragers, which show circadian rhythms, and also in nurses that do not, although the latter have attenuated oscillations in brain mRNA clock gene levels. The oscillations in nurses show that activity can be uncoupled from the circadian network and support the hypothesis that a ticking circadian clock is essential even in around-the-clock active animals in a constant physical environment.

Time-of-day-dependent adaptation of the HPA axis to predictable social defeat stress.

In modern societies, the risk of developing a whole array of affective and somatic disorders is associated with the prevalence of frequent psychosocial stress. Therefore, a better understanding of adaptive stress responses and their underlying molecular mechanisms is of high clinical interest. In response to an acute stressor, each organism can either show passive freezing or active fight-or-flight behaviour, with activation of sympathetic nervous system and the hypothalamus-pituitary-adrenal (HPA) axis providing the necessary energy for the latter by releasing catecholamines and glucocorticoids (GC). Recent data suggest that stress responses are also regulated by the endogenous circadian clock. In consequence, the timing of stress may critically affect adaptive responses to and/or pathological effects of repetitive stressor exposure. In this article, we characterize the impact of predictable social defeat stress during daytime versus nighttime on bodyweight development and HPA axis activity in mice. While 19 days of social daytime stress led to a transient reduction in bodyweight without altering HPA axis activity at the predicted time of stressor exposure, more detrimental effects were seen in anticipation of nighttime stress. Repeated nighttime stressor exposure led to alterations in food metabolization and reduced HPA axis activity with lower circulating adrenocorticotropic hormone (ACTH) and GC concentrations at the time of predicted stressor exposure. Our data reveal a circadian gating of stress adaptation to predictable social defeat stress at the level of the HPA axis with impact on metabolic homeostasis underpinning the importance of timing for the body's adaptability to repetitive stress.

The lateral and dorsal neurons of Drosophila melanogaster: new insights about their morphology and function.

This chapter summarizes our present knowledge about the master clock of the fruit fly at the neuronal level. The clock is organized in distinct groups of interconnected pacemaker neurons with different functions. All of these neurons appear to communicate with one another in order to produce the species-specific activity rhythm, which is organized in morning (M) and evening (E) activity bouts. These two activity components are differentially influenced by distinct groups of pacemaker neurons reminiscent of the Pittendrigh-Daan dual oscillator model. In the original work (Grima et al. 2004; Stoleru et al. 2004), the ventrolateral (LN(v)) and dorsolateral (LN(d)) plus some dorsal groups (DN) of clock neurons have been defined as M and E cells, respectively. We further specify that the clock neurons belong to the M and E oscillators and define a more complex picture of the Drosophila brain clock.

Organization of endogenous clocks in insects.

Insect and mammalian circadian clocks show striking similarities. They utilize homologous clock genes, generating self-sustained circadian oscillations in distinct master clocks of the brain, which then control rhythmic behaviour. The molecular mechanisms of rhythm generation were first uncovered in the fruit fly Drosophila melanogaster, whereas cockroaches were among the first animals where the brain master clock was localized. Despite many similarities, there exist obvious differences in the organization and functioning of insect master clocks. These similarities and differences are reviewed on a molecular and anatomical level.

Neurobiology of the fruit fly's circadian clock.

Studying the fruit fly Drosophila melanogaster has revealed mechanisms underlying circadian clock function. Rhythmic behavior could be assessed to the function of several clock genes that generate circadian oscillations in certain brain neurons, which finally modulate behavior in a circadian manner. This review outlines how individual circadian pacemaker neurons in the fruit fly's brain control rhythm in locomotor activity and eclosion.

The regulation of circadian clocks by light in fruitflies and mice.

A circadian clock has no survival value unless biological time is adjusted (entrained) to local time and, for most organisms, the profound changes in the light environment provide the local time signal (zeitgeber). Over 24 h, the amount of light, its spectral composition and its direction change in a systematic way. In theory, all of these features could be used for entrainment, but each would be subject to considerable variation or 'noise'. Despite this high degree of environmental noise, entrained organisms show remarkable precision in their daily activities. Thus, the photosensory task of entrainment is likely to be very complex, but fundamentally similar for all organisms. To test this hypothesis we compare the photoreceptors that mediate entrainment in both flies and mice, and assess their degree of convergence. Although superficially different, both organisms use specialized (employing novel photopigments) and complex (using multiple photopigments) photoreceptor mechanisms. We conclude that this multiplicity of photic inputs, in highly divergent organisms, must relate to the complex sensory task of using light as a zeitgeber.

The circadian clock of fruit flies is blind after elimination of all known photoreceptors.

Circadian rhythms are entrained by light to follow the daily solar cycle. We show that Drosophila uses at least three light input pathways for this entrainment: (1) cryptochrome, acting in the pacemaker cells themselves, (2) the compound eyes, and (3) extraocular photoreception, possibly involving an internal structure known as the Hofbauer-Buchner eyelet, which is located underneath the compound eye and projects to the pacemaker center in the brain. Although influencing the circadian system in different ways, each input pathway appears capable of entraining circadian rhythms at the molecular and behavioral level. This entrainment is completely abolished in glass(60j) cry(b) double mutants, which lack all known external and internal eye structures in addition to being devoid of cryptochrome.

Drosophila CRY is a deep brain circadian photoreceptor.

cry (cryptochrome) is an important clock gene, and recent data indicate that it encodes a critical circadian photoreceptor in Drosophila. A mutant allele, cry(b), inhibits circadian photoresponses. Restricting CRY expression to specific fly tissues shows that CRY expression is needed in a cell-autonomous fashion for oscillators present in different locations. CRY overexpression in brain pacemaker cells increases behavioral photosensitivity, and this restricted CRY expression also rescues all circadian defects of cry(b) behavior. As wild-type pacemaker neurons express CRY, the results indicate that they make a striking contribution to all aspects of behavioral circadian rhythms and are directly light responsive. These brain neurons therefore contain an identified deep brain photoreceptor, as well as the other circadian elements: a central pace-maker and a behavioral output system.

Ectopic expression of the neuropeptide pigment-dispersing factor alters behavioral rhythms in Drosophila melanogaster.

To study the function of the neuropeptide pigment-dispersing factor (PDF) in the circadian system of Drosophila, we misexpressed the pdf gene from the grasshopper Romalea in the CNS of Drosophila and investigated the effect of this on behavioral rhythmicity. pdf was either ectopically expressed in different numbers of neurons in the brain or the thoracical nervous system or overexpressed in the pacemaker neurons alone. We found severe alterations in the activity and eclosion rhythm of several but not all lines with ectopic pdf expression. Only ectopic pdf expression in neurons that projected into the dorsal central brain severely influenced activity rhythms. Therefore, we conclude that PDF acts as a neuromodulator in the dorsal central brain that is involved in the rhythmic control of behavior. Overexpression of pdf in the pacemaker neurons alone or in the other neurons that express the clock genes period (per) and timeless (tim) did not disturb the activity rhythm. Such flies still showed a rhythm in PDF accumulation in the central brain terminals. This rhythm was absent in the terminals of neurons that expressed PDF ectopically. Probably, PDF is rhythmically processed, transported, or secreted in neurons expressing per and tim, and additional PDF expression in these cells does not influence this rhythmic process. In neurons lacking per and tim, PDF appears to be continuously processed, leading to a constant PDF secretion at their nerve terminals. This may lead to conflicting signals in the rhythmic output pathway and result in a severely altered rhythmic behavior.

Differential control of morning and evening components in the activity rhythm of Drosophila melanogaster--sex-specific differences suggest a different quality of activity.

The rhythms of locomotor activity of male and virgin or mated female flies were compared in the Drosophila melanogaster wild-type strains CantonS, Berlin, and OregonR. Under light-dark conditions, most flies showed a bimodal activity pattern with a morning peak around lights-on and an evening peak before lights-off. For all strains, a distinct sexual dimorphism was observed in the phase of the morning peak. Males had a significantly earlier morning peak than females and consequently a larger phase angle between morning and evening peak (psi(m, e)). Under constant dark conditions, the morning component merged with the evening component to a unimodal activity band in about half of the flies. In those flies who maintained bimodality, the sex-specific difference in psi(m, e) disappeared. Other sex-specific differences were now apparent: Males showed a shorter free-running period than females, and in two of the three strains, females were more active than males. Morning and evening components seem to contribute to the free-running period. Spontaneous or externally provoked change in psi(m, e) were correlated with period changes. In some flies, the morning and the evening components showed splitting, indicating that they are the output of two different oscillators. The sexual dimorphism in the phase of the morning peak under LD-conditions suggests that the function of activity during morning and evening peak might be different, for example, during the morning peak, males are active to find females. Overall, the results underline the multioscillatory nature of Drosophila's circadian system.

Differential regulation of circadian pacemaker output by separate clock genes in Drosophila.

Regulation of the Drosophila pigment-dispersing factor (pdf) gene products was analyzed in wild-type and clock mutants. Mutations in the transcription factors CLOCK and CYCLE severely diminish pdf RNA and neuropeptide (PDF) levels in a single cluster of clock-gene-expressing brain cells, called small ventrolateral neurons (s-LN(v)s). This clock-gene regulation of specific cells does not operate through an E-box found within pdf regulatory sequences. PDF immunoreactivity exhibits daily cycling, but only within terminals of axons projecting from the s-LN(v)s. This posttranslational rhythm is eliminated by period or timeless null mutations, which do not affect PDF staining in cell bodies or pdf mRNA levels. Therefore, within these chronobiologically important neurons, separate elements of the central pacemaking machinery regulate pdf or its product in novel and different ways. Coupled with contemporary results showing a pdf-null mutant to be severely defective in its behavioral rhythmicity, the present results reveal PDF as an important circadian mediator whose expression and function are downstream of the clockworks.

The 69 bp circadian regulatory sequence (CRS) mediates per-like developmental, spatial, and circadian expression and behavioral rescue in Drosophila.

The period (per) gene is an essential component of the circadian timekeeping mechanism in Drosophila. This gene is expressed in a circadian manner, giving rise to a protein that feeds-back to regulate its own transcription. A 69 bp clock regulatory sequence (CRS) has been identified previously upstream of the period gene. The CRS confers wild-type mRNA cycling when used to drive a lacZ reporter gene in transgenic flies. To determine whether the CRS also mediates proper developmental and spatial expression and behavioral rescue, we used the CRS to drive either lacZ or per in transgenic flies. The results show that the CRS is able to activate expression in pacemaker neuron precursors in larvae and essentially all tissues that normally express per in pupae and adults. The CRS is sufficient to rescue circadian feedback loop function and behavioral rhythms in per01 flies. However, the period of locomotor activity rhythms shortens if a stronger basal promoter is used. This study shows that regulatory elements sufficient for clock-dependent and tissue-specific per expression in larvae, pupae, and adults are present in the CRS and that the period of adult locomotor activity rhythms is dependent, in part, on the overall level of per transcripts.

Organization of the circadian system in insects.

The circadian systems of different insect groups are summarized and compared. Emphasis is placed on the anatomical identification and characterization of circadian pacemakers, as well as on their entrainment, coupling, and output pathways. Cockroaches, crickets, beetles, and flies possess bilaterally organized pacemakers in the optic lobes that appear to be located in the accessory medulla, a small neuropil between the medulla and the lobula. Neurons that are immunoreactive for the peptide pigment-dispersing hormone (PDH) arborize in the accessory medulla and appear to be important components of the optic lobe pacemakers. The neuronal architecture of the accessory medulla with associated PDH-immunoreactive neurons is best characterized in cockroaches, while the molecular machinery of rhythm generation is best understood in fruit flies. One essential component of the circadian clock is the period protein (PER), which colocalizes with PDH in about half of the fruit fly's presumptive pacemaker neurons. PER is also found in the presumptive pacemaker neurons of beetles and moths, but appears to have different functions in these insects. In moths, the pacemakers are situated in the central brain and are closely associated with neuroendocrine functions. In the other insects, neurons associated with neuroendocrine functions also appear to be closely coupled to the optic lobe pacemakers. Some crickets and flies seem to possess central brain pacemakers in addition to their optic lobe pacemakers. With respect to neuronal organization, the circadian systems of insects show striking similarities to the vertebrate circadian system.

Effects of small concentrations of volatile anesthetics on action potential firing of neocortical neurons in vitro.

BACKGROUND: Volatile general anesthetics depress neuronal activity in the mammalian central nervous system and enhance inhibitory Cl- currents flowing across the gamma-aminobutyric acid A (GABA(A)) receptor-ion channel complex. The extent to which an increase in GABA(A)-mediated synaptic inhibition contributes to the decrease in neuronal firing must be determined, because many further effects of these agents have been reported on the molecular level. METHODS: The actions of halothane, isoflurane, and enflurane on the firing patterns of single neurons were investigated by extracellular recordings in organotypic slice cultures derived from the rat neocortex. RESULTS: Volatile anesthetics depressed spontaneous action potential firing of neocortical neurons in a concentration-dependent manner. The estimated median effective concentration (EC50) values were about one half the EC50 values for general anesthesia. In the presence of the GABA(A) antagonist bicuculline (20 microM), the effectiveness of halothane, isoflurane, and enflurane in reducing the discharge rates were diminished by 48-65%, indicating that these drugs act via the GABA(A) receptor. CONCLUSIONS: Together with recent investigations, our results provide evidence that halothane, isoflurane, and enflurane reduced spontaneous action potential firing of neocortical neurons in cultured brain slices mainly by increasing GABA(A)-mediated synaptic inhibition. At concentrations, approximately one half the EC50 for general anesthesia, volatile anesthetics increased overall GABA(A)-mediated synaptic inhibition about twofold, thus decreasing spontaneous action potential firing by half.

Robust circadian rhythmicity of Drosophila melanogaster requires the presence of lateral neurons: a brain-behavioral study of disconnected mutants.

Mutations at the disconnected (disco) locus of Drosophila melanogaster disrupt neural cell patterning in the visual system, leading to the loss of many optic lobe neurons. Drosophila's presumptive circadian pacemaker neurons--the dorsal and ventral lateral neurons--are usually among the missing cells, and most disco flies are behaviorally arrhythmic. In this study, I show that ventral lateral neurons (LNvs) are occasionally present and provoke robust circadian rhythmicity in disco mutants. Of 357 individual disco flies four animals with robust circadian rhythmicity were found. All four retained LNvs together with terminals in the superior protocerebrum. Residual or bi-circadian rhythmicity was found in about 20% of all flies; the remaining flies were completely arrhythmic. One of the flies with residual rhythmicity and two of the arrhythmic flies also had some LNvs stained. However, these flies lacked the LNv fibers in the superior protocerebrum. The results suggest that the presence of single LNvs is sufficient to provoke robust circadian rhythmicity in locomotor activity if the LNv terminals reach the superior protocerebrum. The presence of residual or bi-circadian rhythmicity in 20% of the flies without LNvs indicates that also other cells contribute to the rhythmic control of locomotor activity.

Spatial and temporal expression of the period and timeless genes in the developing nervous system of Drosophila: newly identified pacemaker candidates and novel features of clock gene product cycling.

The circadian timekeeping system of Drosophila functions from the first larval instar (L1) onward but is not known to require the expression of clock genes in larvae. We show that period (per) and timeless (tim) are rhythmically expressed in several groups of neurons in the larval CNS both in light/dark cycles and in constant dark conditions. Among the clock gene-expressing cells there is a subset of the putative pacemaker neurons, the "lateral neurons" (LNs), that have been analyzed mainly in adult flies. Like the adult LNs, the larval ones are also immunoreactive to a peptide called pigment-dispersing hormone. Their putative dendritic trees were found to be in close proximity to the terminals of the larval optic nerve Bolwig's nerve, possibly receiving photic input from the larval eyes. The LNs are the only larval cells that maintain a strong cycling in PER from L1 onward, throughout metamorphosis and into adulthood. Therefore, they are the best candidates for being pacemaker neurons responsible for the larval "time memory" (inferred from previous experiments). In addition to the LNs, a subset of the larval dorsal neurons (DNLs) expresses per and tim. Intriguingly, two neurons of this DNL group cycle in PER and TIM immunoreactivity almost in antiphase to the other DNLs and to the LNs. Thus, the temporal expression of per and tim are regulated differentially in different cells. Furthermore, the light sensitivity associated with levels of the TIM protein is different from that in the heads of adult Drosophila.

Development of pigment-dispersing hormone-immunoreactive neurons in the nervous system of Drosophila melanogaster.

An antiserum against the crustacean pigment-dispersing hormone (PDH) was used to identify PDH-immunoreactive neurons in the developing nervous systems of wild type Drosophila melanogaster and the brain mutant disconnected. Particular attention was paid to a group of PDH-immunoreactive neurons at the anterior margin of the medulla-the pigment-dispersing factor-containing neurons close to the medulla (PDFMe neurons)-that seem to be involved in the control of adult circadian rhythmicity. In adults, this group consists of four to six neurons with large somata (large PDFMe neurons) and of four neurons with small somata (small PDFMe neurons). Both subgroups were usually absent in adults of behaviorally arrhythmic mutants of disconnected. In the wild type, PDH immunoreactivity was seen first in the small PDFMe neurons of 4 hour old first-instar larvae. The small PDFMe neurons were found to persist unchanged into adulthood, whereas the large ones seemed to develop halfway through metamorphosis. Beside the PDFMe neurons, three other clusters of PDH-immunoreactive neurons were stained in the developing nervous systems of Drosophila and are described in detail. Two of them were located in the brain, and the third was located in the abdominal neuromeres of the thoracic nervous system. In the mutant disconnected, the larval and the adult set of PDFMe neurons were absent. The other clusters of PDH-immunoreactive neurons seemed to develop normally. The present results are consistent with the hypothesis that the PDFMe neurons are circadian pacemaker neurons that may control rhythmic processes in larvae, pupae, and adults.

The period clock gene is expressed in central nervous system neurons which also produce a neuropeptide that reveals the projections of circadian pacemaker cells within the brain of Drosophila melanogaster.

The period protein (PER) is a essential component of the circadian clock in Drosophila melanogaster. Although PER-containing pacemaker cells have been previously identified in the brain, the neuronal network that comprises the circadian clock remained unknown. Here it is shown that some PER neurons are also immunostained with an antiserum against the crustacean pigment-dispersing hormone (PDH). This antiserum reveals the entire arborization pattern of these pacemaker cells. The arborizations of these neurons are appropriate for modulation of the activity of many neurons and they might interact with PER-containing glial cells. A putative physiological role of PDH in the circadian system is discussed.

Pigment-dispersing hormone-immunoreactive neurons in the nervous system of wild-type Drosophila melanogaster and of several mutants with altered circadian rhythmicity.

Antisera against the crustacean pigment-dispersing hormone (beta-PDH) were used in immunocytochemical preparations to investigate the anatomy of PDH-immunoreactive neurons in the nervous system of wild-type Drosophila melanogaster and in that of several brain mutants of this species, some of which express altered circadian rhythmicity. In the wild-type and in all rhythmic mutants (small optic lobes, sine oculis, small optic lobes; sine oculis), eight cell bodies at the anterior base of the medulla (PDFMe neurons) exhibit intense PDH-like immunoreactivity. Four of the eight somata are large and four are smaller. The four large PDFMe neurons have wide tangential arborizations in the medulla and send axons via the posterior optic tract to the contralateral medulla. Fibers from the four small PDFMe neurons ramify in the median protocerebrum dorsal to the calyces of the mushroom bodies. Their terminals are adjacent to other PDH-immunoreactive somata (PDFCa neurons) which send axons via the median bundle into the tritocerebrum. The results suggest a possible involvement of the PDFMe neurons in the circadian pacemaking system of Drosophila. The location and size of the PDFMe neurons are identical with those of neurons containing the period protein which is essential for circadian rhythmicity. Changes in the arborizations of the PDFMe neurons in small optic lobes; sine oculis mutants are suited to explain the splitting in the locomotor rhythm of these flies. In the arrhythmic mutant, disconnected, the PDFMe neurons are absent. The arrhythmic mutant per0, however, shows normal PDH immunoreactivity and therefore, does not prevent the expression of PDH-like peptides in these neurons.