Membrane electrical excitability is necessary for the free-running larval Drosophila circadian clock

Drosophila larvae and adult pacemaker neurons both express free-running oscillations of period (PER) and timeless (TIM) proteins that constitute the core of the cell-autonomous circadian molecular clock. Despite similarities between the adult and larval molecular oscillators, adults and larvae differ substantially in the complexity and organization of their pacemaker neural circuits, as well as in behavioral manifestations of circadian rhythmicity. We have shown previously that electrical silencing of adult Drosophila circadian pacemaker neurons through targeted expression of either an open rectifier or inward rectifier K(+) channel stops the free-running oscillations of the circadian molecular clock. This indicates that neuronal electrical activity in the pacemaker neurons is essential to the normal function of the adult intracellular clock. In the current study, we show that in constant darkness the free-running larval pacemaker clock-like that of the adult pacemaker neurons they give rise to-requires membrane electrical activity to oscillate. In contrast to the free-running clock, the molecular clock of electrically silenced larval pacemaker neurons continues to oscillate in diurnal (light-dark) conditions. This specific disruption of the free-running clock caused by targeted K(+) channel expression likely reflects a specific cell-autonomous clock-membrane feedback loop that is common to both larval and adult neurons, and is not due to blocking pacemaker synaptic outputs or disruption of pacemaker neuronal morphology.     

Amplitude of circadian oscillations entrained by 24-h light-dark cycles

An intriguing property of circadian clocks is that their free-running period is not exactly 24h. Using models for circadian rhythms in Neurospora and Drosophila, we determine how the entrainment of these rhythms is affected by the free-running period and by the amplitude of the external light-dark cycle. We first consider the model for Neurospora, in which light acts by inducing the expression of a clock gene. We show that the amplitude of the oscillations of the clock protein entrained by light-dark cycles is maximized when the free-running period is smaller than 24h. Moreover, if the amplitude of the light-dark cycle is very strong, complex oscillations occur when the free-running period is close to 24h. In the model for circadian rhythms in Drosophila, light acts by enhancing the degradation of a clock protein. We show that while the amplitude of circadian oscillations entrained by light-dark cycles is also maximized if the free-running period is smaller than 24h, the range of entrainment is centered around 24h in this model. We discuss the physiological relevance of these results in regard to the setting of the free-running period of the circadian clock.     

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.     

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.     

Circadian clock and prothoracicotropic hormone secretion in relation to the larval-larval ecdysis rhythm of the saturniid Samia cynthia ricini

Observations on the timing of ecdysis and neck ligation experiments on larvae of Samia cynthia ricini under various light-dark conditions show that an endogenous circadian clock controls the timing of larval ecdysis and prothoracicotropic hormone (PTTH) secretion preceding it. The clock, upon reaching a specific phase point, causes the brain to secrete PTTH provided that the brain has acquired the secretory competence. This time may vary, in relation to a previous ecdysis, according to the light-dark conditions by which the clock phase is specifically determined, but is fixed relative to a subsequent ecdysis. Thus, in the case of the ecdysis to the 5th instar, PTTH is secreted [15+nτ] hr (τ: free-running period, slightly less than 24 hr) after the clock has started when the rhythm is free-running, and in the second and third nights of the 4th instar under a photoperiod of 12 hr light and 12 hr dark. Full secretion of ecdysone occurs 6 hr after PTTH secretion and ecdysis ensues 34 hr thereafter to complete the ultimate sequence of ecdysis.     

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.     

Light Cycles Given During Development Affect Freerunning Period of Circadian Locomotor Rhythm of period Mutants in Drosophila melanogaster

We reared wild type (Canton-S) and period mutant flies, i.e., per(S) and per(L), of Drosophila melanogaster in constant darkness, constant light or 24h light dark cycles with various light to dark ratios throughout the development from embryo to early adult. The locomotor activity rhythms of newly eclosed individuals were subsequently monitored in the lighting conditions, in which they had been reared, for several days and then in constant darkness. Circadian rhythms were clearly exhibited in constant darkness even in flies reared in constant light and constant darkness as well as flies reared in light-dark cycles, but the freerunning period differed among groups. The results suggest that the circadian clock is assembled without any cyclical photic information, and that the light influences the developing circadian clock of Drosophila to alter the freerunning period. The effects of light on the rhythm differed in some aspects between per(L) flies and the other two strains. Possible mechanisms through which light affects the developing circadian clock are discussed. Copyright 1997 Elsevier Science Ltd. All rights reserved     

Cyclic presence and absence of conspecifics alters circadian clock phase but does not entrain the locomotor activity rhythm of the fruit fly Drosophila melanogaster

Circadian clocks use a wide range of environmental cues, including cycles of light, temperature, food, and social interactions, to fine-tune rhythms in behavior and physiology. Although social cues have been shown to influence circadian clocks of a variety of organisms including the fruit fly Drosophila melanogaster, their mechanism of action is still unclear. Here, the authors report the results of their study aimed at investigating if daily cycles of presence and absence (PA) of conspecific male visitors are able to entrain the circadian locomotor activity rhythm of male hosts living under constant darkness (DD). The results suggest that PA cycles may not be able to entrain circadian locomotor activity rhythms of Drosophila. The outcome does not change when male hosts are presented with female visitors, suggesting that PA cycles of either sex may not be effective in bringing about stable entrainment of circadian clocks in D. melanogaster. However, in hosts whose clock phase has already been set by light/dark (LD) cycles, daily PA cycles of visitors can cause measurable change in the phase of subsequent free-running rhythms, provided that their circadian clocks are labile. Thus, the findings of this study suggest that D. melanogaster males may not be using cyclic social cues as their primary zeitgeber (time cue) for entrainment of circadian clocks, although social cues are capable of altering the phase of their circadian rhythms.     

Balance of activity between LN(v)s and glutamatergic dorsal clock neurons promotes robust circadian rhythms in Drosophila

Circadian rhythms offer an excellent opportunity to dissect the neural circuits underlying innate behavior because the genes and neurons involved are relatively well understood. We first sought to understand how Drosophila clock neurons interact in the simple circuit that generates circadian rhythms in larval light avoidance. We used genetics to manipulate two groups of clock neurons, increasing or reducing excitability, stopping their molecular clocks, and blocking neurotransmitter release and reception. Our results revealed that lateral neurons (LN(v)s) promote and dorsal clock neurons (DN(1)s) inhibit light avoidance, these neurons probably signal at different times of day, and both signals are required for rhythmic behavior. We found that similar principles apply in the more complex adult circadian circuit that generates locomotor rhythms. Thus, the changing balance in activity between clock neurons with opposing behavioral effects generates robust circadian behavior and probably helps organisms transition between discrete behavioral states, such as sleep and wakefulness.     

Circadian synchronization and rhythmicity in larval photoperception-defective mutants of Drosophila

A single light episode during the first larval stage can set the phase of adult Drosophila activity rhythms, showing that a light-sensitive circadian clock is functional in larvae and is capable of keeping time throughout development. These behavioral data are supported by the finding that neurons expressing clock proteins already exist in the larval brain and appear to be connected to the larval visual system. To define the photoreceptive pathways of the larval clock, the authors investigated circadian synchronization during larval stages in various visual systems and/or cryptochrome-defective strains. They show that adult activity rhythms cannot be entrained by light applied to larvae lacking both cryptochrome and the visual system, although such rhythms were entrained by larval stage-restricted temperature cycles. Larvae lacking either pathway alone were light entrainable, but the phase of the resulting adult rhythm was advanced relative to wild-type flies. Unexpectedly, adult behavioral rhythms of the glass60j and norpAP24 visual system mutants that were entrained in the same conditions were found to be severely impaired, in contrast to those of the wild type. Extension of the entrainment until the adult stage restored close to wild-type behavioral rhythms in the mutants. The results show that both cryptochrome and the larval visual system participate to circadian photoreception in larvae and that mutations affecting the visual system can impair behavioral rhythmicity.     

Chronic ethanol consumption impairs the circadian rhythm of pro-opiomelanocortin and period genes mRNA expression in the hypothalamus of the male rat

Certain psychiatric disorders are known to alter the body's biological rhythms. However, currently, very little information is known about the effect of chronic ethanol administration on the circadian clock or the rhythm of beta-endorphin-containing neurons that participate in the control of the reward and reinforcement of alcohol drinking. Here, we report that administration of ethanol, via a liquid diet paradigm for a period of 2 weeks, abolishes the circadian rhythm of pro-opiomelanocortin mRNA expression of beta-endorphin neurons in the arcuate nucleus of the hypothalamus. The circadian expression of the clock governing rat period genes (rPeriod1 mRNA and rPeriod2 mRNA) in the arcuate nucleus was significantly altered, suggesting that ethanol administration disrupted the internal clock. Moreover, ethanol consumption altered the circadian rhythms of rPeriod2 and rPeriod3 mRNA levels in the suprachiasmatic nucleus, suggesting that ethanol also affected the function of the central pacemaker. Our findings identified the vulnerability of the body's clock machinery and its opioidergic system to chronic alcohol drinking.     

The nuclear receptor unfulfilled is required for free-running clocks in Drosophila pacemaker neurons

An intricate neural circuit composed of multiple classes of clock neurons controls circadian locomotor rhythms in Drosophila. Evidence indicates that the small ventral lateral neurons (s-LNvs, M cells) are the dominant pacemaker neurons that synchronize the clocks throughout the circuit and drive free-running locomotor rhythms. Little is known, however, about the molecular underpinning of this unique function of the s-LNvs. Here, we show that the nuclear receptor gene unfulfilled (unf; DHR51) is required for the function of the s-LNvs. UNFULFILLED (UNF) is rhythmically expressed in the s-LNvs, and unf mutant flies are behaviorally arrhythmic. Knockdown of unf in developing LNvs irreversibly destroys the ability of adult s-LNvs to generate free-running rhythms, whereas depletion of UNF from adult LNvs dampens the rhythms of the s-LNvs only in constant darkness. These temporally controlled LNv-targeted unf knockdowns desynchronize circuit-wide molecular rhythms and disrupt behavioral rhythms. Therefore, UNF is a prerequisite for free-running clocks in the s-LNvs and for the function of the entire circadian circuit.     

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.     

The disconnected visual system mutations in Drosophila melanogaster drastically disrupt circadian rhythms

Mutations at the disconnected (disco) locus in Drosophila melanogaster cause cultures of this insect to eclose in an essentially arrhythmic manner and also nearly eliminate free-running circadian rhythms of locomotor activity. Yet disco mutants are not totally light-insensitive: Whereas they performed very poorly in tests of certain behavioral responses to visual stimuli, they were able to exhibit "forced" periodic locomotor activity under conditions of light-dark cycling. We discuss these results in the context of (1) the dispensability of this insect's external photoreceptors for entrainment of its circadian pacemaker, and (2) possible disco-induced abnormalities in the connections of extraocular photoreceptors to their targets in the central nervous system and/or abnormalities in the targets themselves--which presumably include elements of the fly's circadian clock.     

Circadian control of eclosion: interaction between a central and peripheral clock in Drosophila melanogaster

Drosophila melanogaster display overt circadian rhythms in rest:activity behavior and eclosion. These rhythms have an endogenous period of approximately 24 hr and can adjust or "entrain" to environmental inputs such as light. Circadian rhythms depend upon a functioning molecular clock that includes the core clock genes period and timeless (reviewed in and ). Although we know that a clock in the lateral neurons (LNs) of the brain controls rest:activity rhythms, the cellular basis of eclosion rhythms is less well understood. We show that the LN clock is insufficient to drive eclosion rhythms. We establish that the prothoracic gland (PG), a tissue required for fly development, contains a functional clock at the time of eclosion. This clock is required for normal eclosion rhythms. However, both the PG clock function and eclosion rhythms require the presence of LNs. In addition, we demonstrate that pigment-dispersing factor (PDF), a neuropeptide secreted from LNs, is necessary for the PG clock and eclosion rhythms. Unlike other clocks in the fly periphery, the PG is similar to mammalian peripheral oscillators because it depends upon input, including PDF, from central pacemaker cells. This is the first report of a peripheral clock necessary for a circadian event.     

A Circadian Clock of Drosophila: Effects of Deuterium Oxide and Mutations at the period Locus

Mutations at the period (per) locus (1:1.3; 3B1-2) in Drosophila melanogaster lengthen (per^L), shorten (per⁵), or abolish (per°) overt circadian rhythmi-city. Deuterium oxide lengthens the free-running circadian period. We tested the effects of deuterium on three mutants of the per gene (per⁵ per^L, and per°) and wild-type Drosophila melanogaster (per⁺) to assess interactions. With increasing concentrations of deuterium, the free-running circadian period of locomotor activity rhythms increased. The dose-response was linear in all genotypes tested. With increasing dosages ofdeuterium, circadian rhythms became weaker as evidenced by the signal-to-noise ratio (SNR). Genotype and deuterium changed circadian period length independently and additively, showing no interaction. SNRs for all genotypes converged on a low level as deuterium concentration increased. Deuterium increased life span, except at high concentrations (40 and 50%).     

Circadian pacemaker neurons transmit and modulate visual information to control a rapid behavioral response

Circadian pacemaker neurons contain a molecular clock that oscillates with a period of approximately 24 hr, controlling circadian rhythms of behavior. Pacemaker neurons respond to visual system inputs for clock resetting, but, unlike other neurons, have not been reported to transmit rapid signals to their targets. Here we show that pacemaker neurons are required to mediate a rapid behavior. The Drosophila larval visual system, Bolwig's organ (BO), projects to larval pacemaker neurons to entrain their clock. BO also mediates larval photophobic behavior. We found that ablation or electrical silencing of larval pacemaker neurons abolished light avoidance. Thus, circadian pacemaker neurons receive input from BO not only to reset the clock but also to transmit rapid photophobic signals. Furthermore, as clock gene mutations also affect photophobicity, the pacemaker neurons modulate the sensitivity of larvae to light, generating a circadian rhythm in visual sensitivity.     

Circadian modulation of acute alcohol sensitivity but not acute tolerance in Drosophila

An increased understanding of the factors affecting behavioral and neurological responses to alcohol and alcohol physiology is necessary given the tremendous toll alcohol abuse and alcoholism exert on individuals and society. At the behavioral and molecular levels, the response to alcohol appears remarkably conserved from Drosophila to humans, suggesting that investigations across model species can provide insight into the identification of common modulatory factors. We investigated the interaction between the circadian clock and alcohol sensitivity, alcohol tolerance, and alcohol absorbance in Drosophila melanogaster. Using a loss-of-righting reflex (LoRR) assay, we found that flies exhibit a circadian rhythm in the LoRR, with the greatest sensitivity to alcohol occurring from mid to late night, corresponding to the flies' inactive phase. As predicted, a circadian rhythm in the LoRR was absent in circadian mutant flies and under conditions in which the circadian clock was nonfunctional. Circadian modulation of the response to alcohol was not due to circadian regulation of alcohol absorbance. Similar to other animals, Drosophila develop acute and chronic tolerance to alcohol upon repeat exposures. We found that the circadian clock did not modulate the development of acute alcohol tolerance measured as the difference in sensitivity to alcohol between na?ve and pre-exposed flies. Thus, the circadian clock modulates some, but not all, of the behavioral responses to alcohol exposure, suggesting that specific mechanisms underlie the observed circadian modulation of LoRR rather than global cellular circadian regulation. This study provides valuable new insights in our understanding of the circadian modulation of alcohol-induced behaviors that ultimately could facilitate preventative measures in combating alcohol abuse and alcoholism.     

Effects of exposing Drosophila melanogaster parents to ethanol on expression of vestigial in their progeny

When parental Drosophila melanogaster were chronically exposed at 28 degrees C or 24 degrees C to ethanol during their larval and pupal stages of development, their progeny, produced when parents were 5-16-day-old adults, showed modified expression of vestigial alleles in heterozygous and homozygous combinations. Parental alcohol effects were dependent on parental rearing temperature. We conclude that parental environment (alcohol, temperature) causes heritable but transitory changes in progeny phenotype that are elicited by exposure of germ cells to alcohol.