Larval ethanol exposure alters adult circadian free-running locomotor activity rhythm in Drosophila melanogaster

Alcohol consumption causes disruptions in a variety of daily rhythms, including the sleep-wake cycle. Few studies have explored the effect of alcohol exposure only during developmental stages preceding maturation of the adult circadian clock, and none have examined the effects of alcohol on clock function in Drosophila. This study investigates developmental and behavioral correlates between larval ethanol exposure and the adult circadian clock in Drosophila melanogaster, a well-established model for studying circadian rhythms and effects of ethanol exposure. We reared Drosophila larvae on 0%, 10%, or 20% ethanol-supplemented food and assessed effects upon eclosion and the free-running period of the circadian rhythm of locomotor activity. We observed a dose-dependent effect of ethanol on period, with higher doses resulting in shorter periods. We also identified the third larval instar stage as a critical time for the developmental effects of 10% ethanol on circadian period. These results demonstrate that developmental ethanol exposure causes sustainable shortening of the adult free-running period in Drosophila melanogaster, even after adult exposure to ethanol is terminated, and suggests that the third instar is a sensitive time for this effect.     

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.     

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.     

Temperature synchronization of the Drosophila circadian clock

BACKGROUND: Circadian clocks are synchronized by both light:dark cycles and by temperature fluctuations. Although it has long been known that temperature cycles can robustly entrain Drosophila locomotor rhythms, nothing is known about the molecular mechanisms involved. RESULTS: We show here that temperature cycles induce synchronized behavioral rhythms and oscillations of the clock proteins PERIOD and TIMELESS in constant light, a situation that normally leads to molecular and behavioral arrhythmicity. We show that expression of the Drosophila clock gene period can be entrained by temperature cycles in cultured body parts and isolated brains. Further, we show that the phospholipase C encoded by the norpA gene contributes to thermal entrainment, suggesting that a receptor-coupled transduction cascade signals temperature changes to the circadian clock. We initiated the further genetic dissection of temperature-entrainment and isolated the novel Drosophila mutation nocte, which is defective in molecular and behavioral entrainment by temperature cycles but synchronizes normally to light:dark cycles. CONCLUSIONS: We conclude that temperature synchronization of the circadian clock is a tissue-autonomous process that is able to override the arrhythmia-inducing effects of constant light. Our data suggest that it involves a cell-autonomous signal-transduction cascade from a thermal receptor to the circadian clock. This process includes the function of phospholipase C and the product specified by the novel mutation nocte.     

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 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.     

Modulation of circadian clocks by nutrients and food factors

Daily activity rhythms that are dominated by internal clocks are called circadian rhythms. A central clock is located in the suprachiasmatic nucleus of the hypothalamus, and peripheral clocks are located in most mammalian peripheral cells. The central clock is entrained by light/dark cycles, whereas peripheral clocks are entrained by feeding cycles. The effects of nutrients on the central and peripheral clocks have been investigated during the past decade and much interaction between them has come to light. For example, a high-fat diet prolongs the period of circadian behavior, a ketogenic diet advances the onset of locomotor activity rhythms, and a high-salt diet advances the phase of peripheral molecular clocks. Moreover, some food factors such as caffeine, nobiletin, and resveratrol, alter molecular and/or behavioral circadian rhythms. Here, we review nutrients and food factors that modulate mammalian circadian clocks from the cellular to the behavioral level.     

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     

The Drosophila dCREB2 gene affects the circadian clock

We report the role of dCREB2, the Drosophila homolog of CREB/CREM, in circadian rhythms. dCREB2 activity cycles with a 24 hr rhythm in flies, both in a light:dark cycle and in constant darkness. A mutation in dCREB2 shortens circadian locomotor rhythm in flies and dampens the oscillation of period, a known clock gene. Cycling dCREB2 activity is abolished in a period mutant, indicating that dCREB2 and Period affect each other and suggesting that the two genes participate in the same regulatory feedback loop. We propose that dCREB2 supports cycling of the Period/Timeless oscillator. These findings support CREB's role in mediating adaptive behavioral responses to a variey of environmental stimuli (stress, growth factors, drug addiction, circadian rhythms, and memory formation) in mammals and long-term memory formation and circadian rhythms in Drosophila.     

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.     

Insect circadian rhythms and photoperiodism

Two clock-controlled processes, overt circadian rhythmicity and the photoperiodic induction of diapause, are described in the blow fly,Calliphora vicina and the fruit fly,Drosophila melanogaster. Circadian locomotor rhythms of the adult flies reflect endogenous, self-sustained oscillations with a temperature compensated period. The free-running rhythms become synchronised (entrained) to daily light:dark cycles, but become arrhythmic in constant light above a certain intensity. Some flies show fragmented rhythms (internal desynchronisation) suggesting that overt rhythmicity is the product of a multioscillator (multicellular) system. Photoperiodic induction of larval diapause inC. vicina and of ovarian diapause inD. melanogaster is also based on the circadian system but seems, to involve a separate mechanism at both the molecular and neuronal levels. For both processes in both species, the compound eyes and ocelli are neither essential nor necessary for photic entrainment, and the circadian clock mechanism is not within the optic lobes. The central brain is the most likely site for both rhythm generation and extra-optic photoreception. InD. melanogaster, a group of lateral brain neurons has been identified as important circadian pacemaker cells, which are possibly also photo-sensitive. Similar lateral brain neurons, staining for arrestin, a protein in the phototransduction ‘cascade’ and a selective marker for photoreceptors in both vertebrates and invertebrates, have been identified inC. vicina. Much less is known about the cellular substrate of the photoperiodic mechanism, but this may involve thepars intercerebralis region of the mid-brain.     

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.     

The circadian timekeeping system of Drosophila

Daily rhythms in behavior, physiology and metabolism are controlled by endogenous circadian clocks. At the heart of these clocks is a circadian oscillator that keeps circadian time, is entrained by environmental cues such as light and activates rhythmic outputs at the appropriate time of day. Genetic and molecular analyses in Drosophila have revealed important insights into the molecules and mechanisms underlying circadian oscillator function in all organisms. In this review I will describe the intracellular feedback loops that form the core of the Drosophila circadian oscillator and consider how they are entrained by environmental light cycles, where they operate within the fly and how they are thought to control overt rhythms in physiology and behavior. I will also discuss where work remains to be done to give a comprehensive picture of the circadian clock in Drosophila and likely many other organisms.     

JNK regulates the photic response of the mammalian circadian clock

The posttranslational regulation of mammalian clock proteins has been assigned a time-keeping function, but seems to have more essential roles. Here we show that c-Jun N-terminal kinase (JNK), identified by inhibitor screening of BMAL1 phosphorylation at Ser 520/Thr 527/Ser 592, confers dynamic regulation on the clock. Knockdown of JNK1 and JNK2 abrogates BMAL1 phosphorylation and lengthens circadian period in fibroblasts. Mice deficient for neuron-specific isoform JNK3 have altered behavioural rhythms, with longer free-running period and compromised phase shifts to light. The locomotor rhythms are insensitive to intensity variance of constant light, deviating from Aschoff's rule. Thus, JNK regulates a core characteristic of the circadian clock by controlling the oscillation speed and the phase in response to light.     

A lack of locomotor activity rhythms inDrosophila melanogaster larvae (Diptera: Drosophilidae)

We examined the locomotor activity ofDrosophila melanogaster for the existence of circadian rhythms, using the wild type and two mutants of theperiod (per) gene,per ^o andper ^s. This was accomplished using a newly described apparatus for the recording and measurement of larval path lengths over a 96-h test period. None of the larvae examined exhibited appreciable diel rhythms under cycling conditions of light or temperature. Larvae were also not rhythmic under free-running conditions. Our results suggest that theper gene does not influence an observable locomotor behavioral phenotype in the larval stage of development.     

Circadian orchestration of developmental hormones in the insect, Rhodnius prolixus

This review presents a new perspective on the circadian regulation and functions of insect developmental hormones. In Rhodnius prolixus (Hemiptera), the brain neuropeptide prothoracicotropic hormone (PTTH) is released with a circadian rhythm that is controlled by paired photosensitive clocks in the brain. These clocks comprise the dorsal and lateral PER/TIM clock neurons known to regulate behavioral rhythms in Drosophila. Axons of PTTH and clock cells make close contact. Photosensitive PER/TIM clocks also reside in the paired prothoracic glands (PGs), which generate rhythmic synthesis and release of the ecdysteroid molting hormones. The PG clocks are entrained by both light and PTTH. These four clocks are coupled together by both nerves and hormones into a timing system whose primary regulated output is the circadian rhythm of ecdysteroids in the hemolymph. This complex timing system appears necessary to ensure circadian organization of the gene expression that is induced in target cells by ecdysteroids via circadian cycling of the nuclear ecdysteroid receptor (EcR). This multioscillator system serves to transduce 'the day outside' into endocrine rhythms that orchestrate 'the day inside'. It has many functional similarities with vertebrate circadian systems.