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.     

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 pdf neuropeptide gene mutation and ablation of PDF neurons each cause severe abnormalities of behavioral circadian rhythms in Drosophila

The mechanisms by which circadian pacemaker systems transmit timing information to control behavior are largely unknown. Here, we define two critical features of that mechanism in Drosophila. We first describe animals mutant for the pdf neuropeptide gene, which is expressed by most of the candidate pacemakers (LNv neurons). Next, we describe animals in which pdf neurons were selectively ablated. Both sets of animals produced similar behavioral phenotypes. Both sets entrained to light, but both were largely arrhythmic under constant conditions. A minority of each pdf variant exhibited weak to moderate free-running rhythmicity. These results confirm the assignment of LNv neurons as the principal circadian pacemakers controlling daily locomotion in Drosophila. They also implicate PDF as the principal circadian transmitter.     

Drosophila free-running rhythms require intercellular communication

Robust self-sustained oscillations are a ubiquitous characteristic of circadian rhythms. These include Drosophila locomotor activity rhythms, which persist for weeks in constant darkness (DD). Yet the molecular oscillations that underlie circadian rhythms damp rapidly in many Drosophila tissues. Although much progress has been made in understanding the biochemical and cellular basis of circadian rhythms, the mechanisms that underlie the differences between damped and self-sustaining oscillations remain largely unknown. A small cluster of neurons in adult Drosophila brain, the ventral lateral neurons (LN_vs), is essential for self-sustained behavioral rhythms and has been proposed to be the primary pacemaker for locomotor activity rhythms. With an LN_v-specific driver, we restricted functional clocks to these neurons and showed that they are not sufficient to drive circadian locomotor activity rhythms. Also contrary to expectation, we found that all brain clock neurons manifest robust circadian oscillations of timeless and cryptochrome RNA for many days in DD. This persistent molecular rhythm requires pigment-dispersing factor (PDF), an LN_v-specific neuropeptide, because the molecular oscillations are gradually lost when Pdf⁰¹ mutant flies are exposed to free-running conditions. This observation precisely parallels the previously reported effect on behavioral rhythms of the Pdf⁰¹ mutant. PDF is likely to affect some clock neurons directly, since the peptide appears to bind to the surface of many clock neurons, including the LN_vs themselves. We showed that the brain circadian clock in Drosophila is clearly distinguishable from the eyes and other rapidly damping peripheral tissues, as it sustains robust molecular oscillations in DD. At the same time, different clock neurons are likely to work cooperatively within the brain, because the LN_vs alone are insufficient to support the circadian program. Based on the damping results with Pdf⁰¹ mutant flies, we propose that LN_vs, and specifically the PDF neuropeptide that it synthesizes, are important in coordinating a circadian cellular network within the brain. The cooperative function of this network appears to be necessary for maintaining robust molecular oscillations in DD and is the basis of sustained circadian locomotor activity rhythms.     

Drosophila pacemaker neurons require g protein signaling and GABAergic inputs to generate twenty-four hour behavioral rhythms

Intercellular signaling is important for accurate circadian rhythms. In Drosophila, the small ventral lateral neurons (s-LN(v)s) are the dominant pacemaker neurons and set the pace of most other clock neurons in constant darkness. Here we show that two distinct G protein signaling pathways are required in LN(v)s for 24?hr rhythms. Reducing signaling in LN(v)s via the G alpha subunit Gs, which signals via cAMP, or via the G alpha subunit Go, which we show signals via Phospholipase 21c, lengthens the period of behavioral rhythms. In contrast, constitutive Gs or Go signaling makes most flies arrhythmic. Using dissociated LN(v)s in culture, we found that Go and the metabotropic GABA(B)-R3 receptor are required for the inhibitory effects of GABA on LN(v)s and that reduced GABA(B)-R3 expression in?vivo lengthens period. Although no clock neurons produce GABA, hyperexciting GABAergic neurons disrupts behavioral rhythms and s-LN(v) molecular clocks. Therefore, s-LN(v)s require GABAergic inputs for 24?hr rhythms.     

Autoreceptor control of peptide/neurotransmitter corelease from PDF neurons determines allocation of circadian activity in drosophila

Drosophila melanogaster flies concentrate behavioral activity around dawn and dusk. This organization of daily activity is controlled by central circadian clock neurons, including the lateral-ventral pacemaker neurons (LN(v)s) that secrete the neuropeptide PDF (pigment dispersing factor). Previous studies have demonstrated the requirement for PDF signaling to PDF receptor (PDFR)-expressing dorsal clock neurons in organizing circadian activity. Although LN(v)s also express functional PDFR, the role of these autoreceptors has remained enigmatic. Here, we show that (1) PDFR activation in LN(v)s shifts the balance of circadian activity from evening to morning, similar to behavioral responses to summer-like environmental conditions, and (2) this shift is mediated by stimulation of the Gα,s-cAMP pathway and a consequent change in PDF/neurotransmitter corelease from the LN(v)s. These results suggest another mechanism for environmental control of the allocation of circadian activity and provide new general insight into the role of neuropeptide autoreceptors in behavioral control circuits.     

Drosophila Free-Running Rhythms Require Intercellular Communication

Robust self-sustained oscillations are a ubiquitous characteristic of circadian rhythms. These include Drosophila locomotor activity rhythms, which persist for weeks in constant darkness (DD). Yet the molecular oscillations that underlie circadian rhythms damp rapidly in many Drosophila tissues. Although much progress has been made in understanding the biochemical and cellular basis of circadian rhythms, the mechanisms that underlie the differences between damped and self-sustaining oscillations remain largely unknown. A small cluster of neurons in adult Drosophila brain, the ventral lateral neurons (LN_vs), is essential for self-sustained behavioral rhythms and has been proposed to be the primary pacemaker for locomotor activity rhythms. With an LN_v-specific driver, we restricted functional clocks to these neurons and showed that they are not sufficient to drive circadian locomotor activity rhythms. Also contrary to expectation, we found that all brain clock neurons manifest robust circadian oscillations of timeless and cryptochrome RNA for many days in DD. This persistent molecular rhythm requires pigment-dispersing factor (PDF), an LN_v-specific neuropeptide, because the molecular oscillations are gradually lost when Pdf⁰¹ mutant flies are exposed to free-running conditions. This observation precisely parallels the previously reported effect on behavioral rhythms of the Pdf⁰¹ mutant. PDF is likely to affect some clock neurons directly, since the peptide appears to bind to the surface of many clock neurons, including the LN_vs themselves. We showed that the brain circadian clock in Drosophila is clearly distinguishable from the eyes and other rapidly damping peripheral tissues, as it sustains robust molecular oscillations in DD. At the same time, different clock neurons are likely to work cooperatively within the brain, because the LN_vs alone are insufficient to support the circadian program. Based on the damping results with Pdf⁰¹ mutant flies, we propose that LN_vs, and specifically the PDF neuropeptide that it synthesizes, are important in coordinating a circadian cellular network within the brain. The cooperative function of this network appears to be necessary for maintaining robust molecular oscillations in DD and is the basis of sustained circadian locomotor activity rhythms.     

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.     

Pigment-dispersing factor (PDF) has different effects on Drosophila's circadian clocks in the accessory medulla and in the dorsal brain

The neuropeptide pigment-dispersing factor (PDF) is a key transmitter in the circadian clock of Drosophila melanogaster. Here we studied the rhythmic behavior of neural mutants with modified arborizations of the large PDF neurons. In sine oculis(1) (so(1)) mutants we found a higher density of PDF fibers in the fly's pacemaker center, the accessory medulla. These flies exhibited a significantly longer period (24.6 h) than control flies. When PDF levels were elevated to very high levels in the dorsal brain as true for so(mda) mutants and small optic lobes;so(1) double mutants (sol(1);so( 1)), a short-period component split off the long period in behavioral rhythmicity. The short period became shorter the higher the amount of PDF in this brain region and reached a value of approximately 21 h. The period alterations were clearly dependent on PDF, because so(1);Pdf 01 and so(mda);Pdf 01 double mutants showed a single free-running component with a period similar to Pdf 01 mutants (approximately 22.5 h) and significantly longer than the short period of so(mda) mutants. These observations indicate that PDF feeds back on the clock neurons and changes their period. Obviously, PDF lengthens the period of some clock neurons and shortens that of others.     

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.     

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.     

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.     

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.     

Pigment-dispersing factor affects nocturnal activity rhythms, photic entrainment, and the free-running period of the circadian clock in the cricket gryllus bimaculatus

Pigment-dispersing factor (PDF) is a neuropeptide widely distributed in insect brains and plays important roles in the circadian system. In this study, we used RNA interference to study the role of the pigment-dispersing factor (pdf) gene in regulating circadian locomotor rhythms in the cricket, Gryllus bimaculatus. Injections of pdf double-stranded RNA (dspdf) effectively knocked down the pdf mRNA and PDF peptide levels. The treated crickets maintained the rhythm both under light-dark cycles (LD) and constant darkness (DD). However, they showed rhythms with reduced nocturnal activity with prominent peaks at lights-on and lights-off. Entrainability of dspdf-injected crickets was higher than control crickets as they required fewer cycles to resynchronize to the LD cycles shifted by 6 h. The free-running periods of the dspdf-injected crickets were shorter than those of control crickets in DD. These results suggest that PDF is not essential for the rhythm generation but involved in control of the nocturnality, photic entrainment, and fine tuning of the free-running period of the circadian clock.     

The novel Drosophila tim(blind) mutation affects behavioral rhythms but not periodic eclosion

Circadian clock function depends on the tightly regulated exclusion or presence of clock proteins within the nucleus. A newly induced long-period timeless mutant, tim(blind), encodes a constitutively hypophosphorylated TIM protein. The mutant protein is not properly degraded by light, and tim(blind) flies show abnormal behavioral responses to light pulses. This is probably caused by impaired nuclear accumulation of TIM(BLIND) protein, which we observed in brain pacemaker neurons and photoreceptor cells of the compound eye. tim(blind) encodes two closely spaced amino acid changes compared to the wild-type TIM protein; one of them is within a putative nuclear export signal of TIM. Under constant conditions, tim(blind) flies exhibit 26-hr free-running locomotor rhythms, which are not correlated with a period lengthening of eclosion rhythms and period-luciferase reporter-gene oscillations. Therefore it seems possible that TIM--in addition to its well-established role as core clock factor--functions as a clock output factor, involved in determining the period length of adult locomotor 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.     

Drosophila olfactory response rhythms require clock genes but not pigment dispersing factor or lateral neurons

Odors elicit a number of behavioral responses, including attraction and repulsion in Drosophila. In this study, the authors used a T-maze apparatus to show that wild-type Drosophila melanogaster exhibit a robust circadian rhythm in the olfactory attractive and repulsive responses. These responses were lower during the day and began to rise at early night, peaking at about the middle of the night and then declining thereafter. They were also independent of locomotor activity. The olfactory response rhythms were lost in period or timeless mutant flies (per0, tim0), indicating that clock genes control circadian rhythms of olfactory behavior. The rhythms in olfactory response persisted in the absence of the pigment-dispersing factor neuropeptide or the central pacemaker lateral neurons known to drive circadian patterns of locomotion and eclosion. These results indicate that the circadian rhythms in olfactory behavior in Drosophila are driven by pacemakers that do not control the rest-activity cycle and are likely in the antennae.