Light activates output from evening neurons and inhibits output from morning neurons in the Drosophila circadian clock

Animal circadian clocks are based on multiple oscillators whose interactions allow the daily control of complex behaviors. The Drosophila brain contains a circadian clock that controls rest–activity rhythms and relies upon different groups of PERIOD (PER)–expressing neurons. Two distinct oscillators have been functionally characterized under light-dark cycles. Lateral neurons (LNs) that express the pigment-dispersing factor (PDF) drive morning activity, whereas PDF-negative LNs are required for the evening activity. In constant darkness, several lines of evidence indicate that the LN morning oscillator (LN-MO) drives the activity rhythms, whereas the LN evening oscillator (LN-EO) does not. Since mutants devoid of functional CRYPTOCHROME (CRY), as opposed to wild-type flies, are rhythmic in constant light, we analyzed transgenic flies expressing PER or CRY in the LN-MO or LN-EO. We show that, under constant light conditions and reduced CRY function, the LN evening oscillator drives robust activity rhythms, whereas the LN morning oscillator does not. Remarkably, light acts by inhibiting the LN-MO behavioral output and activating the LN-EO behavioral output. Finally, we show that PDF signaling is not required for robust activity rhythms in constant light as opposed to its requirement in constant darkness, further supporting the minor contribution of the morning cells to the behavior in the presence of light. We therefore propose that day–night cycles alternatively activate behavioral outputs of the Drosophila evening and morning lateral neurons.     

Light Activates Output from Evening Neurons and Inhibits Output from Morning Neurons in the Drosophila Circadian Clock

Animal circadian clocks are based on multiple oscillators whose interactions allow the daily control of complex behaviors. The Drosophila brain contains a circadian clock that controls rest–activity rhythms and relies upon different groups of PERIOD (PER)–expressing neurons. Two distinct oscillators have been functionally characterized under light-dark cycles. Lateral neurons (LNs) that express the pigment-dispersing factor (PDF) drive morning activity, whereas PDF-negative LNs are required for the evening activity. In constant darkness, several lines of evidence indicate that the LN morning oscillator (LN-MO) drives the activity rhythms, whereas the LN evening oscillator (LN-EO) does not. Since mutants devoid of functional CRYPTOCHROME (CRY), as opposed to wild-type flies, are rhythmic in constant light, we analyzed transgenic flies expressing PER or CRY in the LN-MO or LN-EO. We show that, under constant light conditions and reduced CRY function, the LN evening oscillator drives robust activity rhythms, whereas the LN morning oscillator does not. Remarkably, light acts by inhibiting the LN-MO behavioral output and activating the LN-EO behavioral output. Finally, we show that PDF signaling is not required for robust activity rhythms in constant light as opposed to its requirement in constant darkness, further supporting the minor contribution of the morning cells to the behavior in the presence of light. We therefore propose that day–night cycles alternatively activate behavioral outputs of the Drosophila evening and morning lateral neurons.     

A subset of dorsal neurons modulates circadian behavior and light responses in Drosophila

A fundamental property of circadian rhythms is their ability to persist under constant conditions. In Drosophila, the ventral Lateral Neurons (LNvs) are the pacemaker neurons driving circadian behavior under constant darkness. Wild-type flies are arrhythmic under constant illumination, but flies defective for the circadian photoreceptor CRY remain rhythmic. We found that flies overexpressing the pacemaker gene per or the morgue gene are also behaviorally rhythmic under constant light. Unexpectedly, the LNvs do not drive these rhythms: they are molecularly arrhythmic, and PDF--the neuropeptide they secrete to synchronize behavioral rhythms under constant darkness--is dispensable for rhythmicity in constant light. Molecular circadian rhythms are only found in a group of Dorsal Neurons: the DN1s. Thus, a subset of Dorsal Neurons shares with the LNvs the ability to function as pacemakers for circadian behavior, and its importance is promoted by light.     

A temperature-dependent timing mechanism is involved in the circadian system that drives locomotor rhythms in the fruit fly Drosophila melanogaster

The circadian clock of Drosophila melanogaster is thought to include rhythmic expression of period gene. Recent studies suggested, however, that a per-less oscillation is also involved in the regulation of circadian locomotor rhythms. In the present study, we examined the existence and the property of the possible per-less oscillation using arrhythmic clock mutant flies carrying per (01), tim(01), dClk(Jrk) or cyc(01), which lack rhythmic per expression. When temperature cycles consisting of 25 degrees C and 30 degrees C with various periods (T=8-32 hr) were given, wild-type (Canton-S) flies showed locomotor rhythms entrained to temperature cycles over a wide range of period (T=8-32 hr) in constant light (LL) while only to T=24 hr in constant darkness (DD). The mutant flies showed rhythms synchronizing with the given cycle both under LL and DD. In per(01) and tim(01) flies, the phase of a major peak slightly changed dependent on Ts in DD, while it did not in dClk(Jrk) and cyc(01) flies. When they were transferred from a constant temperature to a temperature cycle under DD, several cycles were necessary to establish a clear temperature entrainment in per(01) and tim (01) flies. These results suggest that per(01) and tim(01) flies have a temperature-entrainable weak oscillatory mechanism and that the per-less oscillatory mechanism may require dClk and cyc. In addition, per (01) and tim(01) flies changed from thermoactive in DD to cryoactive in LL, while dClk(Jrk) and cyc(01) flies did not. It is thus suggested that dClk and cyc are also involved in determining the light-associated temperature preference in per(01) and tim(01) flies.     

Fixation and locomotor activity are impaired by inducing tetanus toxin expression in adult Drosophila brain

Visual fixation and locomotor activity are two important behavioral properties utilized by flies when they approach a landmark. Although previous studies in Drosophila have revealed that the mushroom bodies (Mbs) and the central complex (CC) were regulatory centers for these behaviors, the specific neurons involved still remain largely unknown. We tested visual fixation behavior and locomotor activity of flies in a simple choice assay, Buridan's paradigm, using the GAL4/UAS system to express tetanus toxin light chain (TeTxLC) in adult neurons specifically. Although we explored a variety of mushroom body and central complex-labeling lines, we found that only four GAL4 lines (104y-GAL4, 121y-GAL4, 154y-GAL4 and 210y-GAL4) could produce significant defects in fixation as well as decrease locomotor activity following adult induction of TeTxLC. This suggests a more complex circuit is involved in controlling these behaviors than previously thought. Expression patterns of the GAL4 lines in the central nervous system provide the some clues to which neurons might be involved in this neural circuit.     

Involvement of the period gene in developmental time-memory: effect of the perShort mutation on phase shifts induced by light pulses delivered to Drosophila larvae

Phases of circadian locomotor activity rhythms of adult Drosophila reared in constant darkness have been shown to be set by a light stimulus delivered as early as the first-instar larval stage. This implies that a circadian clock functions continuously throughout postembryonic development. The clock genes period (per) and timeless (tim) are expressed cyclically in the larval central nervous system of Drosophila, and daily oscillations of per expression persist throughout metamorphosis in a group of cells, which gives rise to the pacemaker cells underlying locomotor activity rhythms of adults. Therefore, PER and TIM cyclings in these neurons may be responsible for the phenomenon of "larval time-memory." In the absence of any evidence for the involvement of these genes in such a developmental clock, and because circadian-pacemaker functions are underanalyzed in terms of the functions during development, the authors tested the time-memory of a fast-clock period mutant. They show that dark-reared perS mutant individuals as well as wild-type flies can be entrained as larvae and that a brief light pulse given to such entrained larvae can induce phase shifts in animals of either genotype. However, the direction and magnitude of phase shifts were different between wild type and perS, suggesting that a clock under the control of period gene participates in the regulation of developmental time-memory. The authors show that the relevant clock can be entrained by two light input pathways, one involving the phospholipase C encoded by the norpA gene, the other mediated by the blue-light receptor cryptochrome. Phase shifts of molecular oscillations during the larval stage were smaller than those measured by adult behavior, suggesting molecularly transient responses during development.     

Blocking synaptic transmission with tetanus toxin light chain reveals modes of neurotransmission in the PDF-positive circadian clock neurons of Drosophila melanogaster

Circadian locomotor rhythms of Drosophila melanogaster are controlled by a neuronal circuit composed of approximately 150 clock neurons that are roughly classified into seven groups. In the circuit, a group of neurons expressing pigment-dispersing factor (PDF) play an important role in organizing the pacemaking system. Recent studies imply that unknown chemical neurotransmitter(s) (UNT) other than PDF is also expressed in the PDF-positive neurons. To explore its role in the circadian pacemaker, we examined the circadian locomotor rhythms of pdf-Gal4/UAS-TNT transgenic flies in which chemical synaptic transmission in PDF-positive neurons was blocked by expressed tetanus toxin light chain (TNT). In constant darkness (DD), the flies showed a free-running rhythm, which was similar to that of wild-type flies but significantly different from pdf null mutants. Under constant light conditions (LL), however, they often showed complex rhythms with a short period and a long period component. The UNT is thus likely involved in the synaptic transmission in the clock network and its release caused by LL leads to arrhythmicity. Immunocytochemistry revealed that LL induced phase separation in TIMELESS (TIM) cycling among some of the PDF-positive and PDF-negative clock neurons in the transgenic flies. These results suggest that both PDF and UNT play important roles in the Drosophila circadian clock, and activation of PDF pathway alone by LL leads to the complex locomotor rhythm through desynchronized oscillation among some of the clock neurons.     

Noncircadian regulation and function of clock genes period and timeless in oogenesis of Drosophila melanogaster

Circadian clock genes are ubiquitously expressed in the nervous system and peripheral tissues of complex animals. While clock genes in the brain are essential for behavioral rhythms, the physiological roles of these genes in the periphery are not well understood. Constitutive expression of the clock gene period was reported in the ovaries of Drosophila melanogaster; however, its molecular interactions and functional significance remained unknown. This study demonstrates that period (per) and timeless (tim) are involved in a novel noncircadian function in the ovary. PER and TIM are constantly expressed in the follicle cells enveloping young oocytes. Genetic evidence suggests that PER and TIM interact in these cells, yet they do not translocate to the nucleus. The levels of TIM and PER in the ovary are affected neither by light nor by the lack of clock-positive elements Clock (Clk) and cycle (cyc). Taken together, these data suggest that per and tim are regulated differently in follicle cells than in clock cells. Experimental evidence suggests that a novel fitness-related phenotype may be linked to noncircadian expression of clock genes in the ovaries. Mated females lacking either per or tim show nearly a 50% decline in progeny, and virgin females show a similar decline in the production of mature oocytes. Disruption of circadian mechanism by either the depletion of TIM via constant light treatment or continuous expression of PER via GAL4/UAS expression system has no adverse effect on the production of mature oocytes.     

Two oscillators are better than one: a circadian pacemaker escapes from the light

Continuous light exposure can suppress circadian rhythms. In this issue of Neuron, Murad et al. demonstrate that, under certain genetic conditions, a novel cluster of pacemaker neurons can drive rhythmic behavior in constant light. Surprisingly, these neurons are distinct from those thought to drive rhythms in constant darkness.     

Circadian regulation of egg-laying behavior in fruit flies Drosophila melanogaster

Significant progress has been made in our understanding of the neurogenetics of circadian clocks in fruit flies Drosophila melanogaster. Several pacemaker neurons and clock genes have now been identified and their roles in the cellular and molecular clockwork established. Some recent findings suggest that the basic architecture of the clock is multi-oscillatory; the clock mechanisms in the ventral lateral neurons (LN(v)s) of the fly brain govern locomotor activity and adult emergence rhythms, while the peripheral oscillators located in antennal cells regulate olfactory rhythm. Among circadian phenomena exhibited by Drosophila, the egg-laying rhythm is unique in many ways: (i) this rhythm persists under constant light (LL), while locomotor activity and adult emergence become arrhythmic, (ii) its circadian periodicity is much longer than 24h, and (iii) while egg-laying is rhythmic under constant darkness, the expression of two core clock genes period (per) and timeless (tim), is non-oscillatory in the ovaries. In this paper, we review our current knowledge of the circadian regulation of egg-laying behavior in Drosophila, and provide some possible explanations for its self-sustained nature. We conclude by discussing the existing limitations in our understanding of the regulatory mechanisms and propose few approaches to address them.     

Targeted expression of tetanus toxin reveals sets of neurons involved in larval locomotion in Drosophila

The Drosophila larva is widely used for studies of neuronal development and function, yet little is known about the neuronal basis of locomotion in this model organism. Drosophila larvae crawl over a plain substrate by performing repetitive waves of forward peristalsis alternated by brief episodes of head swinging and turning. To identify sets of central and peripheral neurons required for the spatial or temporal pattern of larval locomotion, we blocked neurotransmitter release from defined populations of neurons by targeted expression of tetanus toxin light chain (TeTxLC) with the GAL4/UAS system. One hundred fifty GAL4 lines were crossed to a UAS-TeTxLC strain and a motion-analysis system was used to identify larvae with abnormal movement patterns. Five lines were selected that show discrete locomotor defects (i.e., increased turning and pausing) and these defects are correlated with diverse sets of central neurons. One line, 4C-GAL4, caused an unusual circling behavior that is correlated with approximately 200 neurons, including dopaminergic and peptidergic interneurons. Expression of TeTxLC in all dopaminergic and serotonergic but not in peptidergic neurons, caused turning deficits that are similar to those of 4C-GAL4/TeTxLC larvae. The results presented here provide a basis for future genetic studies of motor control in the Drosophila larva.     

Drosophila cryb mutation reveals two circadian clocks that drive locomotor rhythm and have different responsiveness to light

Cryptochrome (CRY) is a blue-light-absorbing protein involved in the photic entrainment of the circadian clock in Drosophila melanogaster. We have investigated the locomotor activity rhythms of flies carrying cryb mutant and revealed that they have two separate circadian oscillators with different responsiveness to light. When kept in constant light conditions, wild-type flies became arrhythmic, while cryb mutant flies exhibited free-running rhythms with two rhythmic components, one with a shorter and the other with a longer free-running period. The rhythm dissociation was dependent on the light intensities: the higher the light intensities, the greater the proportion of animals exhibiting the two oscillations. External photoreceptors including the compound eyes and the ocelli are the likely photoreceptors for the rhythm dissociation, since rhythm dissociation was prevented in so1;cryb and norpAP41;cryb double mutant flies. Immunohistochemical analysis demonstrated that the PERIOD expression rhythms in ventrally located lateral neurons (LNvs) occurred synchronously with the shorter period component, while those in the dorsally located per-expressing neurons showed PER expression most likely related to the longer period component, in addition to that synchronized to the LNvs. These results suggest that the Drosophila locomotor rhythms are driven by two separate per-dependent clocks, responding differentially to constant light.     

A novel circadianly expressed Drosophila melanogaster gene dependent on the period gene for its rhythmic expression.

The Drosophila melanogaster period (per) gene is required for expression of endogenous circadian rhythms of locomotion and eclosion. per mRNA is expressed with a circadian rhythm that is dependent on Per protein; this feedback loop has been proposed to be essential to the central circadian pacemaker. This model would suggest the Per protein also controls the circadian expression of other genetic loci to generate circadian behavior and physiology. In this paper we describe Dreg-5, a gene whose mRNA is expressed in fly heads with a circadian rhythm nearly identical to that of the per gene. Dreg-5 mRNA continues to cycle in phase with that of per mRNA in conditions of total darkness and also when the daily feeding time is altered. Like per mRNA, Dreg-5 mRNA is not expressed rhythmically in per null mutant flies. Dreg-5 encodes a novel 298 residue protein and Dreg-5 protein isoforms also oscillate in abundance with a circadian rhythm. The phase of Dreg-5 protein oscillation, however, is different from that of Per protein expression, suggesting that Dreg-5 and per have common translational but different post-translational control mechanisms. These results demonstrate that the per gene is capable of modulating the rhythmic expression of other genes; this activity may form the basis of the output of circadian rhythmicity in Drosophila.     

Conserved regions of the timeless (tim) clock gene in Drosophila analyzed through phylogenetic and functional studies.

Circadian (approximately 24-hr) rhythms in Drosophila melanogaster depend upon cyclic expression of the period (per) and timeless (tim) genes, which encode interacting components of the endogenous clock. The per gene has been isolated from other insects and, more recently, a per ortholog was found in mammals where its expression oscillates in a circadian fashion. We report here the complete sequence of a tim gene from another species, Drosophila virilis. TIM is better conserved than the PER protein is between these two species (76 vs. 54% overall amino acid identity), and putative functional domains, such as the PER interaction domains and the nuclear localization signal, are highly conserved. The acidic domain and the cytoplasmic localization domain, however, are within the least conserved regions. In addition, the initiating methionine in the D. virilis gene lies downstream of the proposed translation start for the original D. melanogaster tim cDNA and corresponds to the one used by D. simulans and D. yakuba. Among the most conserved parts of TIM is a region of unknown function near the N terminus. We show here that deletion of a 32 amino acid segment within this region affects rescue of rhythms in arrhythmic tim01 flies. Flies carrying a full-length tim transgene displayed rhythms with approximately 24-hr periods, indicating that a fully functional clock can be restored in tim01 flies through expression of a tim transgene. Deletion of the segment mentioned above resulted in very long activity rhythms with periods ranging from 30.5 to 48 hr.     

Old flies have a robust central oscillator but weaker behavioral rhythms that can be improved by genetic and environmental manipulations

Sleep-wake cycles break down with age, but the causes of this degeneration are not clear. Using a Drosophila model, we addressed the contribution of circadian mechanisms to this age-induced deterioration. We found that in old flies, free-running circadian rhythms (behavioral rhythms assayed in constant darkness) have a longer period and an unstable phase before they eventually degenerate. Surprisingly, rhythms are weaker in light-dark cycles and the circadian-regulated morning peak of activity is diminished under these conditions. On a molecular level, aging results in reduced amplitude of circadian clock gene expression in peripheral tissues. However, oscillations of the clock protein PERIOD (PER) are robust and synchronized among different clock neurons, even in very old, arrhythmic flies. To improve rhythms in old flies, we manipulated environmental conditions, which can have direct effects on behavior, and also tested a role for molecules that act downstream of the clock. Coupling temperature cycles with a light-dark schedule or reducing expression of protein kinase A (PKA) improved behavioral rhythms and consolidated sleep. Our data demonstrate that a robust molecular timekeeping mechanism persists in the central pacemaker of aged flies, and reducing PKA can strengthen behavioral rhythms.