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.     

Circadian photoreception in Drosophila: functions of cryptochrome in peripheral and central clocks.

In Drosophila melanogaster, disruption of night by even short light exposures results in degradation of the clock protein TIMELESS (TIM), leading to shifts in the fly molecular and behavioral rhythms. Several lines of evidence indicate that light entrainment of the brain clock involves the blue-light photoreceptor cryptochrome (CRY). In cryptochrome-depleted Drosophila (cry(b)), the entrainment of the brain clock by short light pulses is impaired but the clock is still entrainable by light-dark cycles, probably due to light input from the visual system. Whether cryptochrome and visual transduction pathways play a role in entrainment of noninnervated, directly photosensitive peripheral clocks is not known and the subject of this study. The authors monitored levels of the clock protein TIM in the lateral neurons (LNs) of larval brains and in the renal Malpighian tubules (MTs) of flies mutant for the cryptochrome gene (cry(b)) and in mutants that lack signaling from the visual photopigments (norpA(P41)). In cry(b) flies, light applied during the dark period failed to induce degradation of TIM both in MTs and in LNs, yet attenuated cycling of TIM was observed in both tissues in LD. This cycling was abolished in LNs, but persisted in MTs, of norpA(P41);cry(b) double mutants. Furthermore, the activity of the tim gene in the MTs of cry(b) flies, reported by luciferase, seemed stimulated by lights-on and suppressed by lights-off, suggesting that the absence of functional cryptochrome uncovered an additional light-sensitive pathway synchronizing the expression of TIM in this tissue. In constant darkness, cycling of TIM was abolished in MTs; however, it persisted in LNs of cry(b) flies. The authors conclude that cryptochrome is involved in TIM-mediated entrainment of both central LN and peripheral MT clocks. Cryptochrome is also an indispensable component of the endogenous clock mechanism in the examined peripheral tissue, but not in the brain. Thus, although neural and epithelial cells share the core clock mechanism, some clock components and light-entrainment pathways appear to have tissue-specific roles.     

Cis-combination of the classic per(S) and per(L) mutations results in arrhythmic Drosophila with ectopic accumulation of hyperphosphorylated PERIOD protein

The 1st circadian "clock" gene identified was the X-linked period (per) gene in Drosophila melanogaster. In the pioneering initial report, Konopka and Benzer (1971) characterized 3 alleles of per that shortened (per (S); approximately 19 h), lengthened (per (L); approximately 29 h), or abolished (per (0)) circadian behavioral rhythms. They also showed that transheterozygotes carrying the per (S) and per (L) mutations exhibit robust behavioral rhythms with nearly normal periods of approximately 23 h, highlighting the semidominant nature of many clock mutants. In this study, per (0) flies bearing a doubly mutated per transgene that carries both the per (S) and per (L) alleles (per (0); per (S/L)) were analyzed for behavioral and molecular rhythms. Unlike singly mutated versions, the per (0);per ( S/L) transgenic flies are arrhythmic in constant dark conditions and exhibit little, if any, entrainment to daily light-dark cycles. In a wildtype per (+) background, expression of per ( S/L) abolishes behavioral rhythms, indicating that it functions in a transdominant negative fashion. Biochemical analysis of head extracts revealed that only hyperphosphorylated isoforms of the PERS/L protein are detected throughout a daily cycle, and the levels remain constant. Intriguingly, little if any PERS/L is observed in key pacemaker neurons that control daily activity rhythms, consistent with the notion that hyperphosphorylated isoforms of PER are unstable. Nonetheless, PERS/L is detected in ectopic cells in the brain, in which it exhibits an unusual localization, mainly staining the periphery of the nucleus. These results suggest that posttranslational mechanisms play a key role in limiting the accumulation of PER to specific cells. On a broader scope, our results indicate that the semidominant effects of period-altering alleles observed in trans are not necessarily preserved in the cis-configuration and that novel phenotypes can emerge.     

Widespread receptivity to neuropeptide PDF throughout the neuronal circadian clock network of Drosophila revealed by real-time cyclic AMP imaging

The neuropeptide PDF is released by sixteen clock neurons in Drosophila and helps maintain circadian activity rhythms by coordinating a network of approximately 150 neuronal clocks. Whether PDF acts directly on elements of this neural network remains unknown. We address this question by adapting Epac1-camps, a genetically encoded cAMP FRET sensor, for use in the living brain. We find that a subset of the PDF-expressing neurons respond to PDF with long-lasting cAMP increases and confirm that such responses require the PDF receptor. In contrast, an unrelated Drosophila neuropeptide, DH31, stimulates large cAMP increases in all PDF-expressing clock neurons. Thus, the network of approximately 150 clock neurons displays widespread, though not uniform, PDF receptivity. This work introduces a sensitive means of measuring cAMP changes in a living brain with subcellular resolution. Specifically, it experimentally confirms the longstanding hypothesis that PDF is a direct modulator of most neurons in the Drosophila clock network.     

Genetic and Physiological Characteristics of a Slow-Growing Circadian Clock Mutant of NEUROSPORA CRASSA

A circadian clock mutant of Neurospora crassa with a period length of about 25.8 hours (4 hr longer than wild type) has been isolated after mutagenesis of the band strain. This mutant, called frq-5, segregates as a single nuclear gene, maps near the centromere on linkage group III, and is unlinked to four previously described clock mutants clustered on linkage group VII R (Feldman and Hoyle 1973, 1976). frq-5 differs from the other clock mutants in at least two other respects: (1) it is recessive in heterokaryons, and (2) it grows at about 60% the rate of the parent band strain on both minimal and complete media. Double mutants between frq-5 and each of the other clock mutants show additivity of period length--two long period mutants produce a double mutant whose period length is longer than either of the two single mutants, while a long and a short period double mutant has an intermediate period length. Although slow growth and long periodicity of frq-5 have segregated together among more than 300 progeny, slow growth per se is not responsible for the long period, since all the double mutants have the slow growth characteristic of frq-5, but have period lengths both shorter and longer than wild type.     

cDNA cloning and nuclear localization of the circadian neuropeptide designated as pigment-dispersing factor PDF in the cricket Gryllus bimaculatus

Pigment-dispersing factor (PDF) was recently reported to be a principal circadian neuromodulator involved in transmitting circadian rhythms of daily locomotion in insects. In Drosophila, PDF functions in some of the neurons expressing the clock genes period, timeless, Clock, and cycle, and those clock genes in turn regulate pdf gene expression. In the present study, we cloned a cDNA encoding PDF in the brain of a nocturnal insect, the cricket Gryllus bimaculatus, and found that an isolated clone (310 bp) codes for an extraordinarily short precursor protein with no definite signal sequence, but a nuclear localization signal (NLS)-like sequence instead. The cricket PDF exhibits high sequence identity (78-94%) and similarity (89-100%) to insect PDFs and also to crustacean beta-PDH peptides. In the optic lobes of G. bimaculatus there are PDF-immunoreactive neurons in both the medulla and lamina neuropiles. Among the strongly immunoreactive lamina PDF neurons, on electron microscopy we identified cells exhibiting distinct staining that is not only cytoplasmic but also nuclear. When GFP-fused PDF precursor proteins were expressed in COS-7 cells, distinct translocation of the fusion protein into the nucleus was observed. This is the first finding of PDF peptide in the nucleus, which suggests a fundamental role of PDF peptide per se in the circadian clock system.     

PDF cycling in the dorsal protocerebrum of the Drosophila brain is not necessary for circadian clock function

In Drosophila, the neuropeptide pigment-dispersing factor (PDF) is a likely circadian molecule, secreted by central pacemaker neurons (LNvs). PDF is expressed in both small and large LNvs (sLNvs and lLNvs), and there are striking circadian oscillations of PDF staining intensity in the small cell termini, which require a functional molecular clock. This cycling may be relevant to the proposed role of PDF as a synchronizer of the clock system or as an output signal connecting pacemaker cells to locomotor activity centers. In this study, the authors use a generic neuropeptide fusion protein (atrial natriuretic factor-green fluorescent protein [ANF-GFP]) and show that it can be expressed in the same neurons as PDF itself. Yet, ANF-GFP as well as PDF itself does not manifest any cyclical accumulation in sLNv termini in adult transgenic flies. Surprisingly, the absence of detectable PDF cycling is not accompanied by any detectable behavioral pheno-type, since these transgenic flies have normal morning and evening anticipation in a light-dark cycle (LD) and are fully rhythmic in constant darkness (DD). The molecular clock is also not compromised. The results suggest that robust PDF cycling in sLNv termini plays no more than a minor role in the Drosophila circadian system and is apparently not even necessary for clock output function.     

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 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 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.     

Drosophila ebony activity is required in glia for the circadian regulation of locomotor activity

Previous studies suggest that glia may be required for normal circadian behavior, but glial factors required for rhythmicity have not been identified in any system. We show here that a circadian rhythm in Drosophila Ebony (N-beta-alanyl-biogenic amine synthetase) abundance can be visualized in adult glia and that glial expression of Ebony rescues the altered circadian behavior of ebony mutants. We demonstrate that molecular oscillator function and clock neuron output are normal in ebony mutants, verifying a role for Ebony downstream of the clock. Surprisingly, the ebony oscillation persists in flies lacking PDF neuropeptide, indicating it is regulated by an autonomous glial oscillator or another neuronal factor. The proximity of Ebony-containing glia to aminergic neurons and genetic interaction results suggest a function in dopaminergic signaling. We thus suggest a model for ebony function wherein Ebony glia participate in the clock control of dopaminergic function and the orchestration of circadian activity rhythms.     

A G protein-coupled receptor, groom-of-PDF, is required for PDF neuron action in circadian behavior

The neuropeptide Pigment-Dispersing Factor (PDF) plays a critical role in mediating circadian control of behavior in Drosophila. Here we identify mutants (groom-of-PDF; gop) that display phase-advanced evening activity and poor free-running rhythmicity, phenocopying pdf mutants. In gop mutants, a spontaneous retrotransposon disrupts a coding exon of a G protein-coupled receptor, CG13758. Disruption of the receptor is accompanied by phase-advanced oscillations of the core clock protein PERIOD. Moreover, effects on circadian timing induced by perturbation of PDF neurons require gop. Yet PDF oscillations themselves remain robust in gop mutants, suggesting that GOP acts downstream of PDF. gop is expressed most strongly in the dorsal brain in regions that lie in proximity to PDF-containing nerve terminals. Taken together, these studies implicate GOP as a PDF receptor in Drosophila.     

Separate sets of cerebral clock neurons are responsible for light and temperature entrainment of Drosophila circadian locomotor rhythms

The fruit fly Drosophila melanogaster shows a bimodal circadian locomotor rhythm with peaks at lights-on and before lights-off, which are regulated by multiple clocks in the brain. Even under light-dark cycles, the timing of the evening peak is highly dependent on temperature, starting earlier under lower ambient temperature but terminating almost at the same time. In the present study, using behavioral and immunohistochemical assays, the authors show that separate groups of clock neurons, either light-entrainable or temperature-entrainable, form a functional system driving the locomotor rhythm. When subjected to a light cycle combined with a temperature cycle advanced by 6 h relative to the light cycle, the dorsally located neurons (DNs) and lateral posterior neurons (LPNs) shifted their phase of TIMELESS expression, but the laterally located protocerebral neurons (LNs) basically maintained their original phase. Thus, the LNs seem to be preferentially light-entrainable and the DNs and LPNs to be primarily temperature-entrainable. In pdf(01) mutant flies that lack the neuropeptide PDF in the ventral groups of LNs, the onset of the evening peak was greatly advanced even under synchronized light and temperature cycles and was shifted even more than in wild-type flies in response to a 6-h phase shift of the temperature cycle, suggesting that ventral LNs have a strong impact on the phase of the other cells. It seems likely that the 2 sets of clock cells with different entrainability to light and temperature, and the coupling between them, enable Drosophila to keep a proper phase relationship of circadian activity with respect to the daily light and temperature cycles.     

Function of the Shaw potassium channel within the Drosophila circadian clock

Background

In addition to the molecular feedback loops, electrical activity has been shown to be important for the function of networks of clock neurons in generating rhythmic behavior. Most studies have used over-expression of foreign channels or pharmacological manipulations that alter membrane excitability. In order to determine the cellular mechanisms that regulate resting membrane potential (RMP) in the native clock of Drosophila we modulated the function of Shaw, a widely expressed neuronal potassium (K⁺) channel known to regulate RMP in Drosophila central neurons.

Methodology/Principal Findings

We show that Shaw is endogenously expressed in clock neurons. Differential use of clock gene promoters was employed to express a range of transgenes that either increase or decrease Shaw function in different clusters of clock neurons. Under LD conditions, increasing Shaw levels in all clock neurons (LNv, LNd, DN₁, DN₂ and DN₃), or in subsets of clock neurons (LNd and DNs or DNs alone) increases locomotor activity at night. In free-running conditions these manipulations result in arrhythmic locomotor activity without disruption of the molecular clock. Reducing Shaw in the DN alone caused a dramatic lengthening of the behavioral period. Changing Shaw levels in all clock neurons also disrupts the rhythmic accumulation and levels of Pigment Dispersing Factor (PDF) in the dorsal projections of LNv neurons. However, changing Shaw levels solely in LNv neurons had little effect on locomotor activity or rhythmic accumulation of PDF.

Conclusions/Significance

Based on our results it is likely that Shaw modulates pacemaker and output neuronal electrical activity that controls circadian locomotor behavior by affecting rhythmic release of PDF. The results support an important role of the DN clock neurons in Shaw-mediated control of circadian behavior. In conclusion, we have demonstrated a central role of Shaw for coordinated and rhythmic output from 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.     

BLOCKING ENDOCYTOSIS IN DROSOPHILA'S CIRCADIAN PACEMAKER NEURONS INTERFERES WITH THE ENDOGENOUS CLOCK IN A PDF-DEPENDENT WAY

The neuropeptide pigment-dispersing factor (PDF) plays an essential role in the circadian clock of the fruit fly Drosophila melanogaster, but many details of PDF signaling in the clock network are still unknown. We tried to interfere with PDF signaling by blocking the GTPase Shibire in PDF neurons. Shibire is an ortholog of the mammalian Dynamins and is essential for endocytosis of clathrin-coated vesicles at the plasma membrane. Such endocytosis is used for neurotransmitter reuptake by presynaptic neurons, which is a prerequisite of synaptic vesicle recycling, and receptor-mediated endocytosis in the postsynaptic neuron, which leads to signal termination. By blocking Shibire function via overexpression of a dominant negative mutant form of Shibire in PDF neurons, we slowed down the behavioral rhythm by 3?h. This effect was absent in PDF receptor null mutants, indicating that we interfered with PDF receptor-mediated endocytosis. Because we obtained similar behavioral phenotypes by increasing the PDF level in regions close to PDF neurons, we conclude that blocking Shibire did prolong PDF signaling in the neurons that respond to PDF. Obviously, terminating the PDF signaling via receptor-mediated endocytosis is a crucial step in determining the period of behavioral rhythms. (Author correspondence: charlotte.foerster@biologie.uni-regensburg.de)     

Circadian consequence of socio-sexual interactions in fruit flies Drosophila melanogaster

In fruit flies Drosophila melanogaster, courtship is an elaborate ritual comprising chasing, dancing and singing by males to lure females for mating. Courtship interactions peak in the night and heterosexual couples display enhanced nighttime activity. What we do not know is if such socio-sexual interactions (SSI) leave long-lasting after-effects on circadian clock(s). Here we report the results of our study aimed at examining the after-effects of SSI (as a result of co-habitation of males and females in groups) between males and females on their circadian locomotor activity rhythm. Males undergo reduction in the evening activity peak and lengthening of circadian period, while females show a decrease in overall activity. Such after-effects, at least in males, require functional circadian clocks during SSI as loss-of-function clock mutants and wild type flies interacting under continuous light (LL), do not display them. Interestingly, males with electrically silenced Pigment Dispersing Factor (PDF)-positive ventral lateral (LNv) clock neurons continue to show SSI mediated reduction in evening activity peak, suggesting that the LNv clock neurons are dispensable for SSI mediated after-effects on locomotor activity rhythm. Such after-effects in females may not be clock-dependent because clock manipulated females with prior exposure to males show decrease in overall activity, more or less similar to rhythmic wild type females. The expression of SSI mediated after-effects requires a functional olfactory system in males because males with compromised olfactory ability do not display them. These results suggest that SSI causes male-specific, long-lasting changes in the circadian clocks of Drosophila, which requires the presence of functional clocks and intact olfactory ability in males.