InteractiveFly: GeneBrief

Pigment-dispersing factor receptor: Biological Overview | References

BIOLOGICAL OVERVIEW

Circadian clocks modulate timing of sleep/wake cycles in animals; however, the underlying mechanisms remain poorly understood. In Drosophila melanogaster, large ventral lateral neurons (l-LN_v) are known to promote wakefulness through the action of the neuropeptide pigment dispersing factor (PDF), but the downstream targets of PDF signalling remain elusive. In a screen using downregulation or overexpression (OEX) of the gene encoding PDF receptor (pdfr), this study found that a subset of dopaminergic neurons responds to PDF to promote wakefulness during the day. Moreover, this study found that small LN_v (sLN_v) and dopaminergic neurons form synaptic contacts, and PDFR signalling inhibited dopaminergic neurons specifically during day time. It is proposed that these dopaminergic neurons that respond to PDFR signalling are sleep-promoting and that during the day when PDF levels are high, they are inhibited, thereby promoting wakefulness. Thus, this study has identified a novel circadian clock pathway that mediates wake promotion specifically during day time (Potdar, 2018).

Daily cycles in several environmental factors synchronize endogenous circadian clocks which drive rhythmic sleep/wake patterns in many organisms. Homeostatic mechanisms modulate the amount and depth of sleep, and also allow animals to recover from any sleep deprivation they may have incurred. Together, these processes control the timing and occurrence of sleep and wake states, thereby modulating sleep/wake cycles. Since the discovery that sleep behavior of Drosophila melanogaster is similar to mammalian sleep in several aspects, many pathways and neuronal circuits involving sleep homeostat and circadian clocks have been uncovered. Genes such as minisleep (mns) and hyperkinetic (hk) encoding subunits of Shaker potassium channel function in the sleep homeostat. More recently, central complex structures such as dorsal fan-shaped body (FB) and the ellipsoid body (EB) have been shown to function as effector and modulator of the sleep homeostat, respectively. Meanwhile, mutations in core circadian clock genes such as Clock (clk) and Cycle (cyc) have been shown to cause impaired timing of sleep as they tend to become nocturnal. The circadian neuropeptide pigment dispersing factor (PDF) and its receptor (PDFR) are involved in relaying wake-promoting signals from the circadian pacemaker ventral lateral neurons (LN_vs) in response to light input as well as dopamine. While it has been suggested that the EB may be the downstream target of this wake-promoting PDF/PDFR signaling, the evidence in favor of the same is limited (Potdar, 2018).

In the recent past, in the quest to uncover output pathways of the circadian clocks that help in timing of sleep/wake cycles, a few dedicated circuits have been mapped. Most notably, timing of sleep onset at the beginning of night is a function of increased inhibition of wake-promoting large LN_v (l-LN_v) by GABA (Liu, 2014). In contrast, sleep is suppressed at the end of night by the action of PDF on the PDFR+ dorsal neuron 1 (DN1) group of the circadian network that in turn secretes the wake-promoting neuropeptide diuretic hormone 31 (DH31). Furthermore, yet another group showed that DN1s through glutamate modulate day-time siesta and night-time sleep by inhibiting the morning (small LN_v; s-LN_v) and evening (dorsal lateral neurons; LNds) activity controlling circadian neurons. Yet, none of the studies so far have shed light on how circadian neurons may induce wakefulness during the day (Potdar, 2018).

This question was addressed by screening for putative downstream targets of PDFR signaling by altering the levels of pdfr expression in several subsets of neurons - namely, circadian neurons that are known to express pdfr, subsets of mushroom body (MB) neurons that are sleep- or wake-promoting, wake-promoting pars intercerebralis (PI), sleep homeostat EB, and sleep-promoting FB neurons as well as aminergic neuronal groups, most of which are reported to be wake-promoting. Strikingly, this study found that a subset of dopaminergic neurons responds to changes in pdfr expression by changing the levels of day-time sleep, increasing pdfr levels decreases day-time sleep and vice versa. Moreover, PDF+ and dopaminergic neurons were found to form synaptic contacts with one another, along with the possibility of the former inhibiting the latter. Thus, these results uncover a dedicated pathway involving signaling from the PDF+ neurons perhaps to the PPM3 dopaminergic neurons in the regulation of wakefulness during the day (Potdar, 2018).

Dopamine is primarily involved in promoting wakefulness and is known to act on l-LN_v as well as inhibit sleep-promoting dFB to carry out its wake-promoting function. This study has revealed that certain dopamine neurons are in fact sleep-promoting and through the inhibitory action of PDFR signaling, wakefulness gets promoted specifically during the day. additional experiments that use optogenetic techniques can shed more light on whether these dopaminergic neurons promote sleep directly, or indirectly by preventing wakefulness either through a gating mechanism or by a permissive role. Interestingly, a previous study has found that dopamine acts on l-LN_v to promote wakefulness and this study found that PDFR signaling acts on dopamine neurons, suggesting a feed-forward pathway for wake promotion, where dopamine acting on l-LN_v promotes the inhibition of sleep-promoting dopaminergic neurons by PDFR signaling. The identity of dopamine neurons acting on l-LN_v and those responding to PDFR signaling may differ which can be uncovered with additional experiments (Potdar, 2018).

The role of s-LN_v in modulating sleep and wake has been explored in some detail in the recent years. s-LN_v have also been shown to promote sleep via short NPF (sNPF) as well as myoinhibitory peptide (MiP) by inhibiting the wake-promoting l-LN_v. This study shows that PDF+ s-LN_v make synaptic contacts with dopaminergic neurons and that PDFR signaling inhibits the downstream dopaminergic neurons to promote wakefulness during the day. Moreover, this study has shown a secondary role for s-LN_v in modulating wake-promoting effects of l-LN_v. Yet, how this wake-promoting signal which originates in the l-LN_v gets relayed to the s-LN_v is not understood. Furthermore, from the screen it is clear that this function is not mediated via PDFR signaling among the LN_v, as downregulating and overexpressing pdfr in s-LN_v (Clk 9M GAL4 and Pdf GAL4) do not result in any sleep defects. Thus, l-LN_v to s-LN_v wake-promoting signal is independent of PDF while s-LN_v to dopamine wake-promoting signal requires PDFR signaling (Potdar, 2018).

PDFR being a class B1 GPCR utilizes cAMP as its second messenger, although there is evidence for Ca2+ also acting as the second messenger. For most of the functions of PDF including stabilizing core clock proteins such as TIMELESS and PERIOD in different target neurons such as DN1s and s-LN_v, cAMP is the major secondary messenger. Moreover, it is thought that different actions of PDF of slowing and speeding up of morning and evening clock neurons is also mediated by different components of cAMP signaling mechanism. However, this study shows that for the function of regulating wake levels during the day time, PDFR signaling changes levels of intracellular Ca2+ in dopamine neurons with negligible role for cAMP signaling, suggesting a mechanism by which a neuropeptide that has diverse effects on its downstream targets can modulate different functions independently. This study therefore identified a unique subset of downstream targets for PDFR signaling among the dopamine neurons that promote wakefulness depending on time of day (Potdar, 2018).

Interestingly, in this screen it is noted that there are several driver lines which there are significant changes in day-time sleep but with only one type of manipulation of pdfr levels (Clk 4.1M, 30y, 104y, 121y GAL4). This may be due to ineffective downregulation of pdfr achieved through the Pdfr RNAi line with these particular drivers. Given that PDF is a neuropeptide which can have long-range non-synaptic effects, even misexpressing it (104y and 121y GAL4) in different substrates has resulted in altered day-time sleep levels. Because DH31 can also respond to PDFR (Kunst, 2015), it is possible that these effects could be mediated by DH31 binding to misexpressed PDFR. However, this may not be the case as downregulating DH31-receptor in these regions does not cause changes in sleep levels. Thus, it can be concluded that in regions previously not known to express pdfr, misexpression of pdfr can cause sleep level deficits suggesting that PDF can act in regions which are not direct targets yet may lie in the vicinity of LN_v projections (Potdar, 2018).

The role of PDF/PDFR signaling is well-known in synchronizing the free-running molecular rhythms in neurons across the circadian network. PDFR signaling in the 'evening' neurons (LNd and 5th s-LN_v) is important for appropriate phasing of the evening bout of activity in light/dark cycles. While the role of PDF as a wake-signal has been known, this study demonstrates that a subset of dopaminergic neurons is downstream of the PDF/PDFR signaling. While the PDFR expression is not conclusive, it is shown that perhaps one PPM3 neuron per hemisphere may express the PDFR. Additional experiments that more directly test the functional connectivity between dopaminergic neurons and PDF+ neurons, as well as responsiveness of dopaminergic neurons to PDF may result in a clearer picture. Downregulating pdfr in these neurons results in increase of day-time sleep, which is a phenocopy of the sleep behavior of loss-of-function pdfr whole-body mutants. On the other hand, overexpressing pdfr in these neurons leads to decrease of day-time sleep specifically. It was further shown that PDF and dopaminergic neurons make synaptic contacts with each other at the site of the axonal projection of s-LN_v. Moreover, the effect of PDFR signaling on the PPM3 neurons appears to be inhibitory, suggesting that the PDFR+ PPM3 neurons promote sleep. Taken together, it is concluded that wake-promoting LN_v make synaptic connections with sleep-promoting dopaminergic neurons and promote wakefulness specifically during the day time through inhibitory PDFR signaling (Potdar, 2018).

Neuropeptides PDF and DH31 hierarchically regulate free-running rhythmicity in Drosophila circadian locomotor activity

Neuropeptides play pivotal roles in modulating circadian rhythms. Pigment-dispersing factor (PDF) is critical to the circadian rhythms in Drosophila locomotor activity. This study demonstrates that diuretic hormone 31 (DH31) complements PDF function in regulating free-running rhythmicity using male flies. It was determined that Dh31 loss-of-function mutants (Dh31^#51) showed normal rhythmicity, whereas Dh31(^#51);Pdf⁰¹ double mutants exhibited a severe arrhythmic phenotype compared to Pdf-null mutants (Pdf⁰¹). The expression of tethered-PDF or tethered-DH31 in clock cells, posterior dorsal neurons 1 (DN1ps), overcomes the severe arrhythmicity of Dh31(^#51);Pdf⁰¹double mutants, suggesting that DH31 and PDF may act on DN1ps to regulate free-running rhythmicity in a hierarchical manner. Unexpectedly, the molecular oscillations in Dh31(^#51);Pdf⁰¹ mutants were similar to those in Pdf⁰¹ mutants in DN1ps, indicating that DH31 does not contribute to molecular oscillations. Furthermore, a reduction in Dh31 receptor (Dh31r) expression resulted in normal locomotor activity and did not enhance the arrhythmic phenotype caused by the Pdf receptor (Pdfr) mutation, suggesting that PDFR, but not DH31R, in DN1ps mainly regulates free-running rhythmicity. Taken together, this study identifies a novel role of DH31, in which DH31 and PDF hierarchically regulate free-running rhythmicity through DN1ps (Goda, 2019).

This study has demonstrated a novel function of DH31 in regulating Drosophila locomotor activity rhythms. Dh31^#51 mutants maintained a robust free-running rhythm, whereas Dh31^#51;Pdf⁰¹ double-mutant flies exhibited a severe disruption of their free-running rhythm compared to Pdf⁰¹ mutants. These findings suggest that Dh31^#51 mutants maintain a robust free-running rhythm because the primary factor, PDF, can sustain a strong rhythm. ~40% of Pdf⁰¹ single-mutant flies exhibited a preserved rhythmic state, which is because DH31 can partially support free-running rhythmicity. Thus, the severe disruptions of free-running rhythm in Pdf⁰¹ and Dh31^#51 double-mutant flies is likely caused by the loss of both pathways (Goda, 2019).

PDF is secreted from the main circadian neurons, LN_vs, and acts on other clock cells through PDFR to synchronize and maintain robust molecular rhythms. PDF expression from LN_vs in Dh31^#51;Pdf⁰¹ mutants restored rhythmicity, in contrast to tethered-PDF (t-PDF) expression in LN_vs, indicating that an autoreceptor of PDF signals in LN_vs is not sufficient to maintain rhythmicity. Instead, t-PDF expression in DN1ps restored rhythmicity, suggesting that PDF signaling in DN1ps is sufficient to maintain robust free-running rhythmicity. Recently, the responsiveness to PDF was shown to be strongly altered for 24 h via RalA GTPase in sLN_vs28. Therefore, it is expected that the continuous activation of PDFR by t-PDF generates rhythmic downstream signaling in PDFR-expressing neurons (Goda, 2019).

Molecular oscillations in DN1s were strongly dampened in Pdf⁰¹ mutants compared with WT flies. These data are consistent with previous studies in which the molecular oscillations of PER in Pdf⁰¹ mutants held under DD conditions were dampened in DN1s10 and the genetic manipulation of the circadian clocks in PDF-positive cells altered the molecular rhythms in DN1ps. Furthermore, Pdfr expression in DN1ps has been reported to prevent the arrhythmic phenotype in Pdfr⁵³⁰⁴ mutants. These findings support the idea that PDF is secreted from LN_vs and acts on DN1ps to regulate free-running rhythmicity (Goda, 2019).

Furthermore, it was shown that t-DH31 expression in DN1ps rescued the Pdf⁰¹ and Dh31^#51 double-mutant phenotypes, which suggests that DH31 acts on DN1ps to regulate rhythmicity. Although it has been suggested that DH31 release might increase at dawn and that DH31-mRNA expression levels oscillate for 24 h, how t-DH31 expression causes rhythmic behavioral output remains unclear. Because DH31 can modestly activate PDFR in vitro, it cannot be excluded that t-DH31 overexpression might simply activate PDFR in DN1ps instead of the intrinsic PDF signals, thereby restoring locomotor activity rhythms in the flies. However, the rhythmicity of Dh31^#51;Pdf⁰¹ mutants overexpressing t-DH31 in tim-Gal4-expressing neurons or R18H11-Gal4-expressing DN1ps only reached levels similar to that of the Pdf⁰¹ single-mutant flies. Therefore, DH31 likely acts on DN1ps separately from the PDF pathway (Goda, 2019).

Although it has been shown that DH31 is expressed in a subset of DN1ps, DH31 expression using R18H11-Gal4 did not rescue the Pdf⁰¹ and Dh31^#51 double-mutant phenotypes, suggesting that DH31 expression in R18H11-Gal4-expressing neurons is insufficient to maintain rhythmicity. Instead, DH31 is expressed in DN1as and DH31 expression in tim-Gal4-expressing neurons rescued the phenotype, which suggests that DH31 expression in clock neurons maintains rhythmicity. That said, given that DH31 is expressed in nonclock neurons and that tim-Gal4 is expressed in nonclock cells, it cannot be excluded that DH31 expression in nonclock neurons might play a role in rescuing the severe phenotype of Dh31^#51;Pdf⁰¹ mutants. Alternatively, although DH31 expression in LN_vs was not detectable via anti-DH31 antibody staining, a recent RNA-seq analysis detected Dh31 gene expression in both LN_vs and DN1s. Therefore, DH31 expression from LN_vs may potentially act on DN1s to support locomotor activity rhythms (Goda, 2019).

In summary, it is proposed that PDF and DH31 regulate free-running rhythms in a hierarchical fashion in DN1ps. As t-DH31 or t-PDF expression in DN1ps resulted in a similar level of rhythmicity as that observed in flies expressing t-DH31 or t-PDF, respectively, in tim-Gal4-expressing neurons, DN1ps are at least one of the important clock cells that regulate free-running rhythmicity (Goda, 2019).

Given that Dh31^#51;Pdf⁰¹ mutants exhibited severe arrhythmicity in free-running rhythm, it is speculated that the severe arrhythmic phenotype might be a result of abnormal molecular oscillations. However, the molecular oscillations of Dh31^#51;Pdf⁰¹ mutants were similar to those of Pdf⁰¹ mutants. Therefore, the molecular mechanisms by which DH31 regulates free-running rhythms still remain unclear. Importantly, the peak of VRI expression in LNds in Dh31^#51 was at ZT 19, which was delayed compared with those of WT flies and the other mutants. The data suggested that DH31 is involved in the regulation of molecular oscillations in LNds. Because LNds are the evening pacemaker, the delayed VRI oscillations in LNds might be associated with the longer period of free-running rhythm in Dh31^#51 (Goda, 2019).

Recently, the intracellular calcium rhythms in each clock cell were reported to be nonsynchronous and associated with morning and evening peaks in locomotor activity. DH31 signaling may possibly contribute to the downstream output that controls molecular rhythms in pacemaker processes, such as intracellular calcium rhythms. Given that PDF from sLN_vs regulates strong molecular rhythms in DN1ps and generates robust free-running rhythms under constant conditions, DH31 may help maintain vigorous output signals downstream of the molecular clocks in DN1ps (Goda, 2019).

Recently work has shown that both Dh31r^1/Df mutants and flies undergoing Dh31r knockdown in their neurons showed normal rhythmicity in the locomotor activity rhythm. In contrast to Dh31^#51;Pdf⁰¹ double mutants, Pdfr5304;Dh31r^1/Df double mutants did not enhance the arrhythmicity observed in Pdfr single mutants, which suggests that Dh31r does not complement PDFR function; thus, Dh31r does not function as a receptor for DH31 in this context. Given that Dh31r^1/Df flies showed a strong abnormality in the TPR phenotype, it is more likely that Dh31r does not play an important role in locomotor activity rhythms. However, Dh31r^1/Df4 mutants are not null25, and it cannot be excluded that a small amount of residual Dh31r might drive robust locomotor activity rhythms with the PDF pathway (Goda, 2019).

Which receptors might function with DH31 to regulate free-running rhythmicity? Given that DH31 can activate PDFR in vitro, bath applications of DH31 can activate LN_vs via PDFR19 and DH31 can function as a ligand of PDFR in TPR at the onset of night, PDFR may function as a receptors for both DH31 and PDF in the regulation of free-running rhythmicity. However, because the arrhythmicity of Pdfr5304 mutants was not as severe as that of Dh31^#51;Pdf⁰¹ mutants, PDFR does not appear to act as a receptor for DH31 in this context (Goda, 2019).

Both Dh31r and PDFR are class II G-protein coupled receptors (GPCRs), which also include Hector and Diuretic hormone 44 receptors 1 and 2 (DH44R1 and DH44R2, respectively). Interestingly, the DH44R1 and DH44R2 ligand DH44 has been implicated in circadian output circuits. Therefore, although there is no evidence from in vitro or in vivo experiments, these receptors might nevertheless function as receptors for DH31 to regulate free-running rhythmicity (Goda, 2019).

Orchestration of neuropeptides regulates locomotor activity rhythms in species ranging from flies to mammals The orchestration of neuropeptides is critical for regulating circadian clock functions in species that range from flies to mammals. In mammals, several neuropeptides, including vasoactive intestinal polypeptide (VIP), arginine vasopressin (AVP) and neuromedin S (NMS), are expressed in the SCN, which is the center for circadian clock control. The hierarchy of neuropeptide signaling contributes to circadian function in the SCN. Several recent studies in Drosophila have identified the neuropeptides, including ion transport peptide (ITP), neuropeptide F (NPF), allatostatin A, short neuropeptide F, leucokinin and DH44, that regulate locomotor activity and sleep. However, given that DH31 complements the function of PDF in regulating free-running rhythmicity in the same clock cells, DH31 not only serves as one of the neuropeptides that regulates circadian rhythms but also might selectively influence PDF function in the regulation of free-running rhythms. Thus, these findings shed new light on the next steps required to improve understanding of the core neuropeptide regulatory mechanisms involved in the circadian rhythm (Goda, 2019).

Regulation of PDF receptor signaling controlling daily locomotor rhythms in Drosophila

Each day and in conjunction with ambient daylight conditions, neuropeptide PDF regulates the phase and amplitude of locomotor activity rhythms in Drosophila through its receptor, PDFR, a Family B G protein-coupled receptor (GPCR). The in vivo process was studied by which PDFR signaling turns off, by converting as many as half of the 28 potential sites of phosphorylation in its C terminal tail to a non-phosphorylatable residue (alanine). Many such sites are conserved evolutionarily, and their conversion creates a specific behavioral syndrome opposite to loss-of-function phenotypes previously described for pdfr. That syndrome includes increases in the amplitudes of both Morning and Evening behavioral peaks, as well as multi-hour delays of the Evening phase. The precise behavioral effects were dependent on day-length, and most effects mapped to conversion of only a few, specific serine residues near the very end of the protein and specific to its A isoform. Behavioral phase delays of the Evening activity under entraining conditions predicted the phase of activity cycles under constant darkness. The behavioral phenotypes produced by the most severe PDFR variant were ligand-dependent in vivo, and not a consequence of changes to their pharmacological properties, nor of changes in their surface expression, as measured in vitro. The mechanisms underlying termination of PDFR signaling are complex, subject to regulation that is modified by season, and central to a better understanding of the peptidergic modulation of behavior (Li, 2022).

In Drosophila, neuropeptide PDF signaling helps pattern the output of the fly circadian pacemaker network that controls rhythmic daily locomotor activity. Its functions have been compared to those of neuropeptide VIP in regulating mammalian circadian physiology. Historically PDF was first isolated as an active principle (Pigment Dispersing Hormone) that mediates light-dependent dispersion of pigment granules in diverse chromatophores of crustacea. In the insect circadian system, PDF acts for a specified period within each 24 hr cycle, and works in conjunction with environmental light to set phase and amplitude for locomotor activity rhythms that normally occur around dawn and dusk. Each day, the precise times of dusk and of dawn change, which alters the time interval between them. These facts require that the time of PDF signaling, the point when it starts and the point when it stops, must also be adjusted each day to appropriately follow and reflect these daily variations in the light: dark transitions. PDF signaling starts following its release by specific pacemaker neurons, whose period of activity in vivo tracks the dawn in a variety of photoperiodic conditions. A comparable understanding of how PDF receptor signaling normally stops is lacking: this work addresses that mechanism (Li, 2022).

The PDF receptor (PDFR) is a member of the Family B (secretin receptor-like) GPCR group: it is Gs-coupled and its activation elevates cAMP levels in vivo. It regulates different adenylate cyclases (AC) in diverse target pacemakers (Duval, 2012; Duval, 2013), which, through PKA activation, ultimately regulate the pace of the molecular clock through regulation of Timeless (Seluzicki, 2014). PDFR autoreceptor signaling promotes dramatic, daily morphological changes in the axonal terminals of sLNv pacemakers. In addition to its effects on the pace of the molecular pacemaker, PDFR activation also regulates calcium dynamics in subsets of pacemaker neuron groups to help dictate their group-specific, daily phases of activation (in the sLNv, in the 5th sLNv and in subsets of LNd, and DN3 groups). Such target cell-specific delays of PER-dependent neuronal activity illustrate the basis by which the circadian network produces a daily series of staggered phasic, neuronal outputs. Finally, PDF/PDFR signaling is long-lasting: its depression of basal calcium levels in target neurons persists without abatement over many hours. These observations raise fundamental questions regarding the mechanism and the time course by which PDFR signaling diminishes in anticipation of the next day's cycle of signaling (Li, 2022).

The canonical model of GPCR phosphorylation and homologous desensitization features G protein-coupled receptor kinases (GRKs) which associate with activated GPCRs and phosphorylate cytosolic segments, thereby recruiting βarrestin via its β-arrestins uncouple the receptors from G proteins. or enhance receptor endocytosis; they can also serve as signal transducers by recruiting distinct signaling molecules. A second major regulatory mechanism to reduce GPCR signaling is heterologous desensitization, whereby second-messenger-dependent kinases (PKA or PKC) phosphorylate GPCRs. Thus, experiments were to modify evolutionarily-conserved residues in the C terminal tail of PDFR that could conceivably serve as substrates for phosphorylation and subsequent signal termination. The working hypothesis was that, by their actions in vivo, such modified PDF receptors would reveal extended lifetimes of activation (Li, 2022).

Little is known about the mechanisms that underlie normal termination of PDFR signaling. In the small LNv, pdfr levels are higher late in the day than in early morning. Sensitivity to PDF in vivo peaks in the early day and is regulated by the PER-dependent clock, via post-transcriptional mechanisms: the EC50 for PDF responses in identified neurons varies systematically 5-10 fold: as a consequence of RalA action, as a function of time of day, and as a function of seasonality. PDFR signaling is long-lasting: it persists for many hours in vivo, for as long as free peptide ligand is available in the bath. In addition, β-arrestin2-GFP is not efficiently recruited to activated PDFR when the receptor is functionally-expressed in hEK-293T cells. In contrast, each of 13 other Drosophila neuropeptide GPCRs (including two Family B GPCRs, CG8422 and CG17415) efficiently recruit β-arrestin2-GFP, when they are activated by their cognate ligands in that cellular environment (Li, 2022).

This study reports (1) that PDFR is normally phosphorylated in vivo at conserved C terminal residues; (2) that loss of conserved PDFR phosphorylatable sites leads to a behavioral syndrome opposite to loss-of-function pdf and pdfr phenotypes, with effects on both the amplitude and phase of the daily locomotor peaks. This 'gain of function' approach reveals a multi-hour range of potential phases for both the Morning and Evening activity peaks, within which neuropeptide PDF:PDFR signaling normally specifies rhythmic behavior, according to season. In addition, using a structure-function approach, this work identifies specific PDFR sequence elements that are major points at which the duration of receptor activity is regulated, and through which behavior is modulated in season-specific fashion (Li, 2022).

A gain-of-function approach was used to measure neuropeptide GPCR function in vivo. In doing so, attempts were made to learn about the maximal consequences of PDFR GPCR signaling and also learn something about the phosphorylation events that normally control the duration of PDFR's signaling lifetime. Expression of a PDF receptor variant, containing numerous Ala-substitutions of conserved phosphorylatable residues in the C terminal domain (1-7A), fundamentally altered rhythmic locomotor behavior in Drosophila. The changes included increases in the amplitudes of the Morning and/or Evening activity peaks, and delays in the Phase of the Evening peak. Such results are generally consistent with a prediction whereby non-phosphorylatable PDFR variants-those with a potential to increase the duration of PDFR signaling-would produce behavioral actions opposite to those seen in loss-of-function pdf, or pdfr mutant stocks. Accordingly, it is speculated that phosphorylation of the PDFR at these conserved residues normally defines the duration of its active signaling state each day. The longer duration that follows the substitutions of Ala may allow receptor signal strength to also increase by accrual: such an effect could explain the increased amplitude of the Morning and Evening peaks, as seen with expression of some of the variants. In this context, it is acknowledged that modification of GPCRs by phosphorylation does not exclusively lead to signal termination. GRK2-dependent phosphorylation of the smoothened (smo) GPCR follows reception of the Hh signal and helps mediate its signal: phosphorylation leads to Smo activation in both Drosophila and mammalian systems. Thus modification of putative phosphorylation sites on GPCRs (to preclude phosphorylation) will not exclusively promote extended signaling. Therefore the interpretations must correspondingly consider outcomes without a priori assumption of mechanisms (Li, 2022).

PDFR belongs to the Secretin Receptor Family (Family B) of neuropeptide receptors: there is no clear consensus regarding mechanisms of desensitization and internalization for this receptor family. For VPAC2 receptors, phosphorylation and internalization is mediated exclusively by GRK2. Likewise, Drosophila orthologues of Cortocotrophin Releasing Factor receptors (CG8422, DH44-R1) and Calcitonin receptors (CG17415, DH31-R1) are internalized in hEK cells following recruitment of β-arrestin2. In contrast, PDFR, which is also related to the mammalian Calcitonin receptor, is not internalized following exposure to PDF. VPAC2 receptors are also regulated by heterologous receptor signaling: M3 cholinergic receptors via PKC signaling can block VPAC2 phosphorylation, desensitization and internalization. Furthermore, secretin receptors and VPAC1 receptors undergo phosphorylation by GRKs and β-arrestin2-dependent desensitization, but these are not sufficient to facilitate or mediate internalization. Finally, GLP2-Receptor associates with β-arrestin2 via its distal C terminal sequences, but that receptor domain is required neither for GLP2-R desensitization nor its internalization. Thus kinases other than GRKs and effectors other than β-arrestin2 may regulate internalization of diverse Family B receptors (Li, 2022).

Following activation of rhodopsin in the mammalian retina, visual arrestin is recruited with a time constant of < 80 ms; for many Drosophila neuropeptide receptors, β-arrestin2 is recruited within a minute of exposure to ligand. The PDFR GPCR signals over a time base of many hours: could phosphorylation/de-phosphorylation also regulate its activity? The mammalian blue-light sensitive GPCR melanopsin (OPN4) mediates intrinsic light sensitivity in certain classes of retinal ganglion cells (RGCs): melanopsin signaling is distinguished by long latencies, graded responses and sustained RGC depolarization that can outlast the duration of the light stimulus. Melanopsin-expressing RGCs produce exceptionally long time-course integration because that GPCR behaves differently from other opsins in two fundamental ways. Firstly, it undergoes photoequilibration between signaling and silent states: a property that maintains the availability of pigment molecules for activation, termed 'tristability'. Secondly, phosphorylation and β-arrestin recruitment do contribute to the kinetics of melanopsin signaling, but over a long time-course. It has been proposed that the distal portion of the melanopsin C terminal tail creates a steric blockade covering a cluster of specific serine residues situated in more proximal regions. The time required for relief of that blockade dictates the time course of GPCR phosphorylation and therefore delays subsequent desensitization (Li, 2022).

The 1-7A PDFR variant modified the greatest number of phosphorylatable residues in the series that was tested, and typically produced the strongest behavioral effects. Notably, the 6A variant-which modified only two specific Ser residues-produced effects that were nearly identical to those exhibited by flies expressing the 1-7A form. This suggests that much PDFR post-translational modification, that which is capable of restricting its signaling time-course, may be directed to specific phosphorylation sites near the extreme C terminal end. There are two broadly divergent hypotheses to describe the mechanisms by which GPCR phosphorylation promotes desensitization. The first proposes that modification of specific residues have the greatest significance for downstream desensitizing mechanisms: such a mechanism can explain the effects seen with the highly limited 6A PDFR variant. The second hypothesis invokes triggering of a termination processes by the aggregate negative charge accumulated with bulk phosphorylation, regardless of where it might occur along a GPCR's intracellular sequences. For some GPCRs (e.g., OPN4), the evidence supports both models. The current data concerning PDFR desensitization also support both viewpoints and a model is presented (see A model predicting the effects of phosphorylating different sites on the PDFR CT on downstream signaling.). In particular CL6 and CL7 are considered to be the specific residues especially critical in terminating the time course of PDFR signaling. Notably, converting just the single pair of CL6 AAs, or the single pair of CL7 AAs, is enough to generate hours-long delays in the peaks of locomotor rhythms under Light:Dark conditions. It is noted that the splicing event that distinguishes the PDFR-A and PDFR-D isoforms occurs just prior to the position of sequences encoding CL6 and 7, such that the D form lacks these two highly conserved domains (flybase.org/download/sequence/FBgn0260753/FBpp). Other clusters (like CL2-3) also appear to have potential for 'specific' contributions to PDFR regulation. Together, these results speak to the potency of the inferred specific termination mechanisms for PDFR GPCR signaling. In contrast, the evidence for the bulk phosphorylation hypothesis comes from comparison of the 1-4A, versus the 1-5A, 1-6A and 1-7A variants. This series inactivates increasing numbers of phosphorylatable residues, and with it increasingly delayed evening phases in short day conditions are observed. The tandem mass-spectroscopy results, indicating endogenous phosphorylation of certain PDFR residues demonstrates such post-translational modifications can occur. However, it is cautioned that these phospho-peptide measurements derive from whole head extracts and may not provide a complete or relevant accounting of those sites that are modified in critical E pacemaker neurons (Li, 2022).

The results also point to what is proposed as 'context-dependent' effects of modifying specific GPCR residues-ones that may produce opposing behavioral effects, depending on the phosphorylation status of neighboring sites. In particular, opposing results were observed with Ala-variants of the CL5 site. In comparing results from the series 1-4A, 1-5A, 1-6A and 1-7A, it was found that under Short Day conditions, the 1-4A had only mild effects on the evening peak phase compared to over-expressing WT PDFR. Whereas 1-5A, 1-6A and 1-7A all produced significant delays. The difference between the 1-$A and 1-5A variants is a change in a single S residue at CL5 (S633A): Those observations suggest modification of the CL5 residue normally promotes desensitization of PDFR and termination of PDFR signaling. However, the PDFR variant 5-7A was constructed with the expectation that it would be as effective as 1-5A, 1-6A or 1-7A in delaying the evening phase. Instead it proved only weakly effective. This same mis-match of expected behavioral effects for 5-7A was also seen in other photoperiodic conditions. Likewise, Langlet (2005) reported that effects on mutating Serine residues in the terminus of VPAC1 did not produce additive effects. It is proposed that phosphorylation of CL5 will have either positive or negative consequences on PDFR desensitization depending on which other neighboring residues are also modified. Thus it is speculated that CL5 takes on outsize importance in determining desensitization rates for PDFR and so may itself be subject to exceptional regulation. Such complex interactions between phosphorylation sites is reminiscent of interactions documented between diverse phosphorylation sites in the circadian clock protein PERIOD. Better resolution of these two paradoxical mechanisms [(1) specific phosphorylation versus bulk negative charge, and (2) context-dependent effects of CL5 site phosphorylation] awaits precise molecular definition of where and when PDFR is modified in key pacemaker neurons in vivo, and by which post-translational modifications (Li, 2022).

The actions of PDFR variants that were described, following substitutions of Ala for various Ser/Thr or Tyr residues, are not easily explained by a hypothesis invoking neomorphic or constitutive GPCR properties. The evidence for this conclusion is three-fold. First, the effects on both amplitude and phase of activity peaks by these variants are not of a random assortment: rather they are all strictly opposite to that of loss-of-function models for pdf and pdfr. Second, the actions of PDFR variants display strong dependence on wild type pdf gene function: this strongly argues that the actions of PDFR variants reflect responses to normal, endogenous PDF signaling. Third, even the strongest actions of PDFR variants on the phases and amplitudes of locomotor activity (that of the 1-7A variant) reflected PDFR sequence variation, and not the properties of the GFP C-terminal fusion. Together these observations support the hypothesis that phosphorylation of some or all of the C terminal residues that were studied are normally modified to attenuate the strength and duration of PDFR signaling during the 24-hr day (Li, 2022).

PDF neuropeptide actions were discovered in the context of physiological adaptions to daylight in crustacea. PDF expression was discovered within a defined subset of the insect Drosophila circadian pacemaker network. Pdf has been shown to make a fundamental contribution by setting the normal behavioral phase of Evening activity in Drosophila during light:dark entrainment and promoting normal rhythmicity during constant darkness. PDF signaling coordinates with that driven by light: It works in parallel to direct photosensitivity along with the CRY blue light photoreceptor. Flies doubly-mutant for cry and pdf display pronounced deficits of locomotor rhythmicity, and can be as severe as measured in clock-deficient flies. PDF and environmental light also work coordinately at the level of neuronal activity patterns: in the case of the evening pacemakers (LNd and the 5th s-LNv), both light and PDF signaling promote a delay in their PER-dependent activation period, and together help align it to a phase just prior to dusk. Based on the close association of PDF signaling and photoperiodic signaling, it is not surprising that the duration of the photoperiod strongly influenced the effects of PDFR sequence variants on the Morning versus the Evening activity peaks. Delayed Evening activity peak produced by PDFR gain-of-function variants under Short Day conditions has been reported: that delay appears strongly inhibited by light durations > 8 hr. It remains unclear at what molecular level light exerts such an inhibitory effect, however, a similarity is noted of these observations with effects recently reported from loss-of-function states for the phosphatase PRL-1. Like the most active PDFR variants this study described (e.g., 6A, 7A, 1-5A 1-6A and 1-7A), PRL-1 mutants produce a 3-4 h delay in the phase of the evening activity peak, but only under winter-like (short day) conditions, and not under equinox or summer-like (long day) conditions. It has been shown that TIM phosphorylation is affected by PRL-1 activity and suggest the seasonal action of PRL-1 to advance the evening locomotor activity phase is mediated by modification of TIM levels within the transcription-translation feedback loop. The extensive similarity of these two sets of behavioral phenotypes reveals either serial or parallel pathways to effect comparable outcomes on locomotor activity. If serial, then according to the simplest model, PRL1 acts downstream of PDFR, and the combined results to-date suggest PRL-1 would be inhibited by PDFR activation. Further genetic and biochemical experiments are needed to evaluate if and how these pathways converge (Li, 2022).

Using an in vitro assay for cyclic AMP generation, this study found that modifying phosphorylation properties of PDFR does not affect the strength of signaling. That conclusion suggests that in vivo other GPCR features are normally affected to regulate the extent/duration of PDFR signaling according to season. This speculation corresponds to findings described with other Family B GPCRs like VPAC1: Mutation of all the Ser and Thr residues of the C terminal tail, and of Ser250 to Ala, led to a receptor with binding properties and adenylate cyclase activity similar to the wild type receptor; however that variant receptor was neither phosphorylated nor internalized. It is proposed that the PDFR variants this study has described do not modify locomotor behavior by virtue of greater second messenger signaling. Rather they do so by generating cAMP (and perhaps other second messengers) over time periods longer than that normal sustained by a WT receptor over a portion of each 24-hr cycle (Li, 2022).

Is PDFR modulation of diurnal phase independent of its modulation of circadian period? A conventional interpretation of the phenotypes that have been described is that PDFR signaling affects the pace of the molecular clock (re-setting the period) and consequently affects behavioral phase indirectly, which is downstream of the clock. However, PDFR variants reliably delayed the Evening activity phase under winter-like conditions, but these variants did not significantly lengthen circadian period. All effects of the 1-7A variant on circadian period were derived from the GFP fusion. Therefore, an alternative hypothesis cannot be ruled out: that the duration of PDF>PDFR signaling to target pacemakers in the Drosophila brain is modulated each day to directly delay Evening pacemaker neuronal activity independent of its effects on entrainment of the molecular clock. In fact, the pdfr han mutant flies exhibited quasi-normal tau values in this report, although they more traditionally exhibit shortened ones. Evening locomotor activity derives in large part from the activity of the primary Evening oscillators, termed E cells, the LNd and the 5th small LNv. However, numerous network interactions are also known to provide critical contributions to the accuracy, precision and adaptability of that timing system. The Evening oscillator group itself is known to be heterogeneous and to constitute at least three separate, functionally distinct oscillators, only some of which express PDFR-A and respond to PDF. It has been reported that the phase of Period protein accumulation in E neurons is phase-locked to the previous lights-off transition and also sensitive to PDF signaling. It was concluded that the peak of PER accumulation is key to determining the phase of the evening activity peak, but also note that the correlation between the Period-clock timing cue and the Evening activity peak is not perfect. It has been suggested the Evening behavioral phase is better described by also factoring in the delay in E cell neuronal activity driven by PDF neuromodulation. It is speculated that it is the combination of two separate PDFR signaling effects that is essential for proper rhythmic behavioral outcomes across seasons. If valid, the hypothesis predicts a bifurcation of signaling pathways downstream of PDFR, to independently regulate diurnal phase and also circadian period within E pacemaker neurons (Li, 2022).

Neuroprotective effects of PACAP against paraquat-induced oxidative stress in the Drosophila central nervous system

Parkinson's disease (PD) is a progressive neurodegenerative movement disorder that can arise after long-term exposure to environmental oxidative stressors, such as the herbicide paraquat (PQ). This study investigated the potential neuroprotective action of vertebrate pituitary adenylate cyclase-activating polypeptide (PACAP) against PQ in Drosophila. Pretreatment with this neuropeptide applied to the ventral nerve cord (VNC) at low doses markedly extended the survival of wild-type decapitated flies exposed to neurotoxic levels of PQ or dopamine (DA). In contrast and interestingly, application of a PACAP receptor antagonist, PACAP-6-38, had opposite effects, significantly decreasing the resistance of flies to PQ. PACAP also reduced PQ-induced caspase activation and reactive oxygen species (ROS) accumulation in the VNC. This study sought the endogenous neuropeptide receptor potentially involved in PACAP-mediated neuroprotection in Drosophila. Knocking down the gene encoding the receptor PDFR of the neuropeptide pigment-dispersing factor (PDF) in all neurons conferred to flies higher resistance to PQ, whereas PDFR downregulation restricted to PDF or DA neurons did not increase PQ resistance, but remarkably suppressed the neuroprotective action of PACAP. Further experiments performed with Pdf and Pdfr-deficient mutant strains confirmed that PDF and its receptor are required for PACAP-mediated neuroprotection in flies. Evidence using split-GFP reconstitution is provided that PDF neurons make synaptic contacts onto DA neurons in the abdominal VNC. These results, therefore, suggest that the protective action of PACAP against PQ-induced defects in the Drosophila nervous system involves the modulation of PDFR signaling in a small number of interconnected neurons (Hajji, 2019).

On variations in the level of PER in glial clocks of Drosophila optic lobe and its negative regulation by PDF signaling

The level of the core protein of the circadian clock Period (PER) expressed by glial peripheral oscillators depends on their location in the Drosophila optic lobe. It appears to be controlled by the ventral lateral neurons (LN_vs) that release the circadian neurotransmitter Pigment Dispersing Factor (PDF). Glial cells of the distal medulla neuropil (dMnGl) that lie in the vicinity of the PDF-releasing terminals of the LN_vs possess receptors for PDF (PDFRs) and express PER at significantly higher level than other types of glia. Surprisingly, the amplitude of PER molecular oscillations in dMnGl is increased twofold in PDF-free environment, that is in Pdf⁰ mutants. The Pdf⁰ mutants also reveal an increased level of glia-specific protein REPO in dMnGl. The photoreceptors of the compound eye (R-cells) of the PDF-null flies, on the other hand, exhibit de-synchrony of PER molecular oscillations, which manifests itself as increased variability of PER-specific immunofluorescence among the R-cells. Moreover, the daily pattern of expression of the presynaptic protein Bruchpilot (BRP) in the lamina terminals of the R-cells is changed in Pdf⁰ mutant. Considering that PDFRs are also expressed by the marginal glia of the lamina that surround the R-cell terminals, the LN_v pacemakers appear to be the likely modulators of molecular cycling in the peripheral clocks of both the glial cells and the photoreceptors of the compound eye. Consequently, some form of PDF-based coupling of the glial clocks and the photoreceptors of the eye with the central LN_v pacemakers must be operational (Gorska-Andrzejak, 2018).

NMDA receptor-mediated Ca2+ influx in the absence of Mg2+ block disrupts rest:activity rhythms in Drosophila

The correlated activation of pre- and postsynaptic neurons is essential for the NMDA receptor-mediated Ca2+ influx by removing Mg2+ from block site and NMDA receptors have been implicated in phase resetting of circadian clocks. So this study assessed rest:activity rhythms in Mg2+ block defective animals. Using Drosophila locomotor monitoring system, circadian rest:activity rhythms of different mutants were checked under constant darkness (DD) and light:dark (LD) conditions. Mg2+ block defective mutant flies were found to exhibit completely arrhythmic under DD. To further understand the role of Mg2+ block in daily circadian rest:activity, the mutant flies were observed under LD cycles, and severely reduced morning anticipation and advanced evening peak compared to control flies. Tissue-specific expression of Mg2+ block defective NMDA receptors was used, and pigment-dispersing factor receptor (PDFR) expressing circadian neurons were implicated in mediating the circadian rest:activity deficits. Endogenous functional NMDA receptors are expressed in most Drosophila neurons, including in a subgroup of dorsal neurons (DN1s). Subsequently, it was determined that the uncorrelated extra Ca2+ influx may act in part through Ca2+/Calmodulin (CaM)-stimulated PDE1c pathway leading to morning behavior phenotypes. These results demonstrate that Mg2+ block of NMDA receptors at resting potential is essential for the daily circadian rest:activity and it is proposes that Mg2+ block functions to suppress CaM-stimulated PDE1c activation at resting potential, thus regulating Ca2+ and cAMP oscillations in circadian and sleep circuits (Song, 2017).

Drosophila DH31 neuropeptide and PDF receptor regulate night-onset temperature preference

Body temperature exhibits rhythmic fluctuations over a 24 h period and decreases during the night, which is associated with sleep initiation. However, the underlying mechanism of this temperature decrease is largely unknown. Previous work has shown that Drosophila exhibit a daily temperature preference rhythm (TPR), in which their preferred temperatures increase during the daytime and then decrease at the transition from day to night (night-onset). Because Drosophila are small ectotherms, their body temperature is very close to that of the ambient temperature, suggesting that their TPR generates their body temperature rhythm. This study demonstrates that the neuropeptide diuretic hormone 31 (DH31) and pigment-dispersing factor receptor (PDFR) contribute to regulate the preferred temperature decrease at night-onset. PDFR and tethered-DH31 expression in dorsal neurons 2 (DN2s) restore the preferred temperature decrease at night-onset, suggesting that DH31 acts on PDFR in DN2s. Notably, it was previously shown that the molecular clock in DN2s is important for TPR. Although PDF (another ligand of PDFR) is a critical factor for locomotor activity rhythms, Pdf mutants exhibit normal preferred temperature decreases at night-onset. This suggests that DH31-PDFR signaling specifically regulates a preferred temperature decrease at night-onset. Thus, it is proposed that night-onset TPR and locomotor activity rhythms are differentially controlled not only by clock neurons but also by neuropeptide signaling in the brain (Goda, 2016).

Body temperature rhythm (BTR) is fundamental for maintaining homeostasis, such as in generating metabolic energy and sleep. BTR is one of the most robust circadian outputs and can affect the peripheral clocks of mammals. The rhythmic patterns of BTR and locomotor activity rhythms are analogous. For instance, in diurnal mammals, both body temperature and locomotor activity increase during the daytime and decrease at night. Nonetheless, BTR and locomotor activity rhythms are regulated by different subsets of subparaventricular zone (SPZ) neurons, suggesting that these rhythms are controlled independently (Goda, 2016).

Mammals are not the only examples of this phenomenon. Previous work has shown that Drosophila exhibit a daily temperature preference rhythm (TPR), in which their preferred temperatures increase during the daytime and then decrease at the transition from day to night (night-onset) (Kaneko, 2012). Because Drosophila are small ectotherms, their body temperature is very close to that of the ambient temperature, suggesting that their TPR generates their BTR. In Drosophila, TPR and locomotor activity rhythms are regulated by different subsets of clock neurons (Kaneko, 2012). There are ~150 central pacemaker cells in the fly brain, which are functionally homologous to mammalian suprachiasmatic nucleus (SCN) neurons. These pacemaker cells are approximately divided into lateral neurons [LNs; small ventral LNs (s-LN_vs), large ventral LNs (l-LN_vs), and dorsal lateral neurons (LNds)] and dorsal neurons (DNs; DN1, DN2, and DN3) based on their location and size. The clocks in DN2s are critical for regulating TPR during the daytime (Kaneko, 2012), but not for regulating locomotor activity rhythms (Kaneko, 2012; Goda, 2016 and references therein).

Neuropeptides and their receptors have important roles in synchronizing circadian clocks. A class II G-protein-coupled receptor, pigment-dispersing factor receptor (PDFR), and its ligand (PDF) play important roles in synchronizing circadian clocks and are required for robust circadian locomotor activity in Drosophila. Notably, PDF and PDFR function in a similar manner to vasoactive intestinal peptide (VIP) and its receptor VPAC2 in mammals, both of which play important roles in the ability of clock neurons to regulate the rhythmicity and synchrony of both locomotor activity rhythms and BTRs (Goda, 2016).

Recent reports have suggested that, in addition to PDF, diuretic hormone 31 (DH31) also activates PDFR based on in vitro experiments (Mertins, 2005) and a study that used brain imaging with bath-applied DH31 (Shafer, 2008). Moreover, it has been shown that DH31 is expressed in the posterior dorsal neurons 1 (DN1ps) and that it modulates sleep as a wake-promoting signal before dawn but does not affect locomotor activity rhythms in Drosophila (Kunst, 2014). DH31 is a functional homolog of mammalian calcitonin gene-related peptide (CGRP), which mediates thermosensation and thermoregulation. However, it is unknown whether CGRP is involved in the regulation of BTR in mammals (Goda, 2016).

This study demonstrates that DH31 and PDFR play important roles for TPR at night-onset. DN2s are the main clock cells for TPR, and the data suggest that DH31 binding to PDFR in DN2s regulates temperature preference decreases at night-onset, which is the first in vivo evidence that DH31 could function as a ligand of PDFR. Therefore, it is proposed that circadian locomotor activity and night-onset TPR are regulated by different neuropeptides that use the same receptor expressed in different clock cells (Goda, 2016).

Both Dh31 and Pdfr mutants exhibited abnormal night-onset TPR, and t-DH31 and PDFR expression in DN2s are sufficient to control night-onset TPR, suggesting that DH31 could be a ligand of PDFR in DN2s. Unexpectedly, PDF, an important neuropeptide for locomotor activity, is not required for night-onset TPR. It suggests that PDF and DH31 appear to act on different subsets of PDFR-expressing clock cells to regulate locomotor activity rhythms and night-onset TPR, respectively. Together, the data suggest that locomotor activity and night-onset TPR are regulated through PDFR by different subsets of clock neurons using different neuropeptides. Nonetheless, because all Dh31, Pdf, and Pdfr mutants still maintain normal daytime TPR, the underlying molecular mechanisms of daytime TPR are still obscure (Goda, 2016).

In humans, body temperature dramatically decreases at night, which is associated with sleep initiation. Although it suggests a relationship between BTR and sleep-wake cycles, the underlying mechanisms are largely unclear. A recent study suggested that DH31 mediates sleep-wake cycles and that DH31 secretion functions as a wake-promoting signal before dawn (Kunst, 2014). Given that it was found that DH31 is required for night-onset TPR, DH31 could regulate both sleep-wake cycles and TPR with different timing (i.e., at night-onset for TPR and before dawn for sleep) (Goda, 2016).

While t-DH31 expression in DN2s rescues the Dh31#51 phenotype, t-PDF expression in DN2s slightly rescues in comparison with UAS controls. This shows that t-DH31 in DN2s rescues more efficiently than t-PDF in DN2s. However, DH31 stimulates PDFR less efficiently than PDF in vitro (Mertens, 2005). This suggests that either DH31 may more efficiently bind PDFR in DN2s than PDF or that DN2s may express another receptor of DH31 that also regulates night-onset TPR. Notably, although the DH31 receptor (DH31R) is a known receptor for DH31 in vitro, this study observed that DH31R is not expressed in DN2s and that the Dh31r mutant exhibited normal night-onset TPR. Therefore, it would be interesting to further explore whether additional receptors for DH31 in DN2s are involved in night-onset TPR. It has been previously shown that flies in the light prefer a 1°C higher temperature than in the dark (light-dependent temperature preference [LDTP]), thus demonstrating that light influences temperature preference behavior. Importantly, while LDTP is only affected by light, night-onset TPR is affected by both light and time (ZT10-ZT12 and ZT13-ZT15). Therefore, they are not controlled by the same pathway. In fact, PDFR expression using Clk4.5F-Gal4 rescues the abnormal LDTP phenotype of Pdfr5304 mutants, but it did not rescue night-onset TPR (Goda, 2016).

DH31 is a functional homolog of mammalian CGRP. Importantly, CGRP is related to many physiological functions, such as temperature sensation, migraines, and chronic pain in mammals. CGRP has recently attracted attention for its role in the treatment of migraine attacks, which are associated with body-temperature fluctuations. Additionally, CGRP is expressed in the SCN, but its function in circadian rhythms is not entirely clear. Thus, the findings raise the possibility that CGRP may be involved in BTR regulation and may mediate the connection between the circadian clock and other physiological functions. Moreover, this research will not only expand current understanding of neuropeptidergic regulation, but may also ultimately facilitate a discovery of the fundamental mechanisms of BTR (Goda, 2016).

The influence of light on temperature preference in Drosophila

Ambient light affects multiple physiological functions and behaviors, such as circadian rhythms, sleep-wake activities, and development, from flies to mammals. Mammals exhibit a higher body temperature when exposed to acute light compared to when they are exposed to the dark, but the underlying mechanisms are largely unknown. The body temperature of small ectotherms, such as Drosophila, relies on the temperature of their surrounding environment, and these animals exhibit a robust temperature preference behavior. This study demonstrates that Drosophila prefer a ~1° higher temperature when exposed to acute light rather than the dark. This acute light response, light-dependent temperature preference (LDTP), was observed regardless of the time of day, suggesting that LDTP was regulated separately from the circadian clock. However, screening of eye and circadian clock mutants suggested that the circadian clock neurons posterior dorsal neurons 1 (DN1ps) and Pigment-Dispersing Factor Receptor (PDFR) play a role in LDTP. To further investigate the role of DN1ps in LDTP, PDFR in DN1ps was knocked down, resulting in an abnormal LDTP. The phenotype of the pdfr mutant was rescued sufficiently by expressing PDFR in DN1ps, indicating that PDFR in DN1ps was responsible for LDTP. These results suggest that light positively influences temperature preference via the circadian clock neurons, DN1ps, which may result from the integration of light and temperature information. Given that both Drosophila and mammals respond to acute light by increasing their body temperature, the effect of acute light on temperature regulation may be conserved evolutionarily between flies and humans (Head, 2015)

PDF and cAMP enhance Per stability in Drosophila clock neurons

The neuropeptide PDF is important for Drosophila circadian rhythms: pdf⁰¹ (pdf-null) animals are mostly arrhythmic or short period in constant darkness and have an advanced activity peak in light-dark conditions. PDF contributes to the amplitude, synchrony, as well as the pace of circadian rhythms within clock neurons. PDF is known to increase cAMP levels in PDR receptor (PDFR)-containing neurons. However, there is no known connection of PDF or of cAMP with the Drosophila molecular clockworks. This study discovered that the mutant period gene per^S ameliorates the phenotypes of pdf-null flies. The period protein (Per) is a well-studied repressor of clock gene transcription, and the per^S protein (PerS) has a markedly short half-life. The result therefore suggests that the PDF-mediated increase in cAMP might lengthen circadian period by directly enhancing Per stability. Indeed, increasing cAMP levels and cAMP-mediated protein kinase A (PKA) activity stabilizes Per, in S2 tissue culture cells and in fly circadian neurons. Adding PDF to fly brains in vitro has a similar effect. Consistent with these relationships, a light pulse causes more prominent Per degradation in pdf⁰¹ circadian neurons than in wild-type neurons. The results indicate that PDF contributes to clock neuron synchrony by increasing cAMP and PKA, which enhance Per stability and decrease clock speed in intrinsically fast-paced PDFR-containing clock neurons. It is further suggested that the more rapid degradation of PerS bypasses PKA regulation and makes the pace of clock neurons more uniform, allowing them to avoid much of the asynchrony caused by the absence of PDF (Li, 2014).

Since the original observation that pdf⁰¹ flies have a highly reliable 1-2 h advanced activity phase in LD and short period in DD before they become arrhythmic, it has been assumed that PDF functions at least in part to lengthen the period of at least some brain oscillators that run too fast in its absence. Indeed, there is evidence in favor of this notion, and it is likely that the pdf⁰¹ strain arrhythmicity results from conflicts between neuronal oscillators that run too fast and others that maintain a ∼24-h pace or may even run more slowly without PDF. The substantial improvement of pdf⁰¹ rhythmicity by the per^S gene therefore suggests that per^S endows all oscillators with such a short period that they have a more uniform pace and substantially reduced oscillator asynchrony without PDF (Li, 2014).

Although there was no information on how PDF might function to lengthen the period of the fast oscillators, the effect of per^S implicates Per as a candidate molecular target. Because PerS is known to disappear rapidly in the nighttime, this further suggests that the Per degradation rate might be the biochemical target of PDF period lengthening. An even more specific version of this notion follows from the PDF-mediated increases in cAMP levels in PDFR-expressing clock neurons. Because PDFR is expressed in many clock neurons, including subsets of LN_vs, LNds, and DN1s, this increase in cAMP may slow the pace of Per degradation in intrinsically fast-paced PDFR-expressing clock neurons. Indeed, the data indicate that increasing cAMP levels and PKA activity inhibits Per degradation in cell culture as well as in fly brains. Although these increases are probably in excess of what normally occurs in response to PDF, addition of PDF to brains in vitro has a similar effect. Because the additions of kinase inhibitors Rp-cAMPS and PKI increased the rate of Per degradation in S2 cells as well as in brains, it is suggested that PDF-induced up-regulation of cAMP level and PKA activity likely affect Per stability (Li, 2014).

A light pulse at night caused more prominent Per degradation in pdf⁰¹ mutant flies than in wild-type flies. As nighttime light also causes premature Tim degradation and a consequent advance in Per degradation in many clock neurons, some of these neurons could be the intrinsically fast (22- to 23-h period) oscillators that are impacted by PDF and experience enhanced cAMP levels to slow their rate of Per degradation and clock pace. These probably include the s-LN_vs and the DN1s, many of which are PDFR-positive. Based on the behavioral phenotype of pdf⁰¹ flies in LD and DD, the effect of PDF on Per degradation probably occurs in the late night-early morning in a LD cycle and at the same (subjective) time in DD. This is also the time when Per degradation is most prominent (Li, 2014).

Interestingly, the firing rate of PDF-containing neurons, the l-LN_vs as well as the s-LN_vs, is also maximal near the beginning of the day, in DD as well as LD; this is also the likely time of maximal PDF release from s-LN_v dorsal projections. In addition, the l-LN_vs promote light-mediated arousal, also mediated at least in part by PDF. Taken together with the fact that light has been shown to increase the firing rate of l-LN_vs in a CRY-dependent manner, it is likely that lights on in the morning also potentiates the PDF-cAMP system. Note that the end of the night-beginning of the day is the time in the circadian cycle dominated by clock protein turnover, i.e., this is when there is little per or tim RNA or protein synthesis. This further supports a focus on clock protein turnover regulation at these times (Li, 2014).

Because the mammalian neuropeptide VIP contributes to oscillator synchrony within the SCN in a manner that resembles at least superficially the contribution of PDF to oscillator synchrony within the fly brain circadian network, VIP might function similarly to PDF. However, VIP probably connects differently to the mammalian clock system. For example, morning light almost certainly up-regulates clock protein transcription in mammals, for example, per1 transcription. Therefore, VIP-mediated up-regulation of cAMP levels probably activates CREB and clock gene transcription through CRE sites in mammalian clock gene promoters rather than influencing clock protein turnover like in flies (Li, 2014).

The stabilization effect of PDF and cAMP on Per requires PKA activity within circadian neurons. The effect could be indirect, through unknown PKA targets including other clock proteins. However, Per is known to be directly phosphorylated by multiple kinases; they include Nemo, which stabilizes Per. In addition, a study in Neurospora shows that PKA directly phosphorylates and stabilizes FRQ. Because FRQ and Per have similar roles, protein turnover in the two clock systems may be similar beyond the shared role of the CK1 kinase. Based also on the S2 cell experiments, it is suggested that PKA directly phosphorylates Per and enhances its stability. This could occur by inhibiting a conformational switch to a less stable structure, a possibility that also applies to NEMO-mediated Per stabilization. PKA could also phosphorylate other clock proteins; this is by analogy to the known Per kinases Nemo and Doubletime (Dbt), which also phosphorylate Clk (Li, 2014).

The more rapid intrinsic degradation of PerS may at least partially bypass the effect of PKA phosphorylation and therefore PDFR stimulation. This may endow all circadian neurons with a more uniform period, which can maintain synchrony and therefore rhythmicity without PDF. The fact that PerS is less sensitive than Per to increases in cAMP levels is consistent with this interpretation, although an earlier phase of PerS degradation might also influence this result (Li, 2014).

One further consideration is the 0.5-h period difference between the per^S and the per^S;;pdf⁰¹ strains. A residual period-lengthening effect of PDF suggests that per^S does not endow all oscillators with the identical period, i.e., that there is still some asynchrony between different per^S neurons without PDF. This may reflect an incomplete bypass of PKA by PerS or an additional effect of cAMP or PKA on other clock proteins. Nonetheless, several per^S neuronal oscillators maintain a strong amplitude without PDF. Although this is commonly taken to reflect an effect on synchrony, another possibility is based on data indicating that PDF normally enhances oscillator amplitude as well as synchrony; weak amplitudes may then be the more proximal cause of behavioral arrhythmicity. With this notion in mind, it is suggested that PerS-containing oscillators are not only short period but also more robust, i.e., that the more rapid turnover of PerS makes the clock stronger. More robust rhythmicity is also apparent in the behavioral records of all per^S-containing strains. In this view, the stronger degradation 'drive' of PerS makes these oscillators more cell autonomous and therefore less dependent on neuronal mechanisms like firing and PDF release, which enhance oscillator synchrony and amplitude. The general notion is that discrete differences in clock molecule properties can change the relationship of the transcriptional cycle to the circadian brain network (Li, 2014).

The Drosophila neuropeptides PDF and sNPF have opposing electrophysiological and molecular effects on central neurons

Neuropeptides have widespread effects on behavior, but how these molecules alter the activity of their target cells is poorly understood. A new model system was employed in Drosophila to assess the electrophysiological and molecular effects of neuropeptides, recording in situ from larval motor neurons which transgenically express a receptor of choice. Focus was placed on two neuropeptides, Pigment-dispersing factor (PDF) and short neuropeptide F (sNPF), which play important roles in sleep/rhythms and feeding/metabolism. PDF treatment depolarized motor neurons expressing the PDF receptor (PDFR), increasing excitability. sNPF treatment had the opposite effect, hyperpolarizing neurons expressing the sNPF receptor (sNPFR). Live optical imaging using a genetically encoded FRET-based sensor for cyclic AMP (cAMP) showed that PDF induced a large increase in cAMP, whereas sNPF caused a small but significant decrease in cAMP. Co-expression of pertussis toxin or RNAi interference to disrupt the G-protein Galphao blocked the electrophysiological responses to sNPF, showing that sNPFR acts via Galphao signaling. Using a fluorescent sensor for intracellular calcium, it was observed that sNPF-induced hyperpolarization blocked spontaneous waves of activity propagating along the ventral nerve cord, demonstrating that the electrical effects of sNPF can cause profound changes in natural network activity in the brain. This new model system provides a platform for mechanistic analysis of how neuropeptides can affect target cells at the electrical and molecular level, allowing for predictions of how they regulate brain circuits that control behaviors such as sleep and feeding (Vecsey, 2014).

Calcitonin gene-related peptide neurons mediate sleep-specific circadian output in Drosophila

Imbalances in amount and timing of sleep are harmful to physical and mental health. Therefore, the study of the underlying mechanisms is of great biological importance. Proper timing and amount of sleep are regulated by both the circadian clock and homeostatic sleep drive. However, very little is known about the cellular and molecular mechanisms by which the circadian clock regulates sleep. This study describes a novel role for diuretic hormone 31 (DH31), the fly homolog of the vertebrate neuropeptide calcitonin gene-related peptide, as a circadian wake-promoting signal that awakens the fly in anticipation of dawn. RESULTS: Analysis of loss-of-function and gain-of-function Drosophila mutants demonstrates that DH31 suppresses sleep late at night. DH31 is expressed by a subset of dorsal circadian clock neurons that also express the receptor for the circadian neuropeptide pigment-dispersing factor (PDF). PDF secreted by the ventral pacemaker subset of circadian clock neurons acts on PDF receptors in the DH31-expressing dorsal clock neurons to increase DH31 secretion before dawn. Activation of PDF receptors in DH31-positive DN1 specifically affects sleep and has no effect on circadian rhythms, thus constituting a dedicated locus for circadian regulation of sleep. This study has identified a novel signaling molecule (DH31) as part of a neuropeptide relay mechanism for circadian control of sleep. These results indicate that outputs of the clock controlling sleep and locomotor rhythms are mediated via distinct neuronal pathways (Kunst, 2014).

Vertebrate Calcitonin gene-related peptide (CGRP) has been implicated in controlling anxietyand stress response. While not previously addressed experimentally, the intimate relationship between stress, anxiety, and sleep suggests that CGRP might regulate sleep. Potentially relevant to this possibility is the recent observation that acute activation of CGRP signaling in zebrafish larvae increases spontaneous locomotor activity and decreases quiescence. This analysis of gain-of-function and loss-of-function mutant flies establishes that DH31, the Drosophila homolog of CGRP, is a negative regulator of sleep maintenance that awakens the animal in anticipation of dawn. This finding motivates investigation of a potential role for CGRP in the regulation of vertebrate sleep (Kunst, 2014).

Using powerful tools for cell-specific neuronal manipulation, this study identified a highly restricted subset of DN1 circadian clock neurons that secrete DH31 late at night to awaken the fly in anticipation of dawn. Neuropeptides secreted from the circadian pacemaker in the mammalian SCN, such as prokinecticin 2 and cardiotrophin-like cytokine, are important clock outputs, but their cellular targets and molecular mechanisms remain unknown. In flies, PDF-expressing sLNv pacemaker neurons are known to be upstream of DN1s, sLNvs are most active around dawn, and PDF secretion from LNvs suppresses sleep at night. However, how PDF signals propagate out of the circadian clock network to regulate sleep remains unknown (Kunst, 2014).

This study shows that PDF secreted by the sLNv pacemaker neurons activates PDFR in the DH31-expressing DN1s to increase neuronal activity and DH31 secretion late at night, thereby awakening the fly in anticipation of dawn. This is consistent with an earlier report demonstrating that flies lacking PDF or PDFR sleep more late at night. However, unlike DH31, PDF also promotes wake during the day. This suggests that PDF regulation of daytime sleep is mediated by neurons other than the DH31-expressing DN1s. PDFR is expressed by circadian clock neurons in addition to DN1s, and PDF signaling to these other clock neurons could be responsible for the wake-promoting effect of PDF during the day (Kunst, 2014).

While PDF plays a key role in circadian timekeeping, neither constitutive activation of PDFR in the DH31-expressing DN1s nor manipulation of their secretion of DH31 affects free-running circadian rhythms. This establishes the PDF-to-DH31 neuropeptide relay as a novel sleep-specific output of the circadian pacemaker network, independent from and parallel to the outputs that drive circadian rhythms themselves. Since the basic cellular and molecular organization of vertebrate and insect circadian networks is conserved, this motivates the search for similar mechanisms in the SCN. Future studies are required to identify the neuronal targets of sleep-regulating DH31 signals and the cellular and molecular mechanisms by which DH31-R1 activation in these targets induces wake (Kunst, 2014).

Differentially timed extracellular signals synchronize pacemaker neuron clocks

Synchronized neuronal activity is vital for complex processes like behavior. Circadian pacemaker neurons offer an unusual opportunity to study synchrony as their molecular clocks oscillate in phase over an extended timeframe (24 h). To identify where, when, and how synchronizing signals are perceived, the minimal clock neural circuit in Drosophila larvae were studied, manipulating either the four master pacemaker neurons (LN_vs) or two dorsal clock neurons (DN1s). Unexpectedly, it was found that the PDF Receptor (PdfR) is required in both LN_vs and DN1s to maintain synchronized LN_v clocks. It was also found that glutamate is a second synchronizing signal that is released from DN1s and perceived in LN_vs via the metabotropic glutamate receptor (mGluRA). Because simultaneously reducing Pdfr and mGluRA expression in LN_vs severely dampened Timeless clock protein oscillations, it is concluded that the master pacemaker LN_vs require extracellular signals to function normally. These two synchronizing signals are released at opposite times of day and drive cAMP oscillations in LN_vs. Finally it was found that PdfR and mGluRA also help synchronize Timeless oscillations in adult s-LN_vs. It is proposed that differentially timed signals that drive cAMP oscillations and synchronize pacemaker neurons in circadian neural circuits will be conserved across species (Collins, 2014).

PDF neuron firing phase-shifts key circadian activity neurons in Drosophila

This study addressed two long-standing models for the function of the Drosophila brain circadian network: a dual oscillator model, which emphasizes the primacy of PDF-containing neurons, and a cell-autonomous model for circadian phase adjustment. Five different circadian (E) neurons were identified that are a major source of rhythmicity and locomotor activity. Brief firing of PDF cells at different times of day generates a phase response curve (PRC), which mimics a light-mediated PRC and requires PDF receptor expression in the five E neurons. Firing also resembles light by causing TIM degradation in downstream neurons. Unlike light however, firing-mediated phase-shifting is CRY-independent and exploits the E3 ligase component CUL-3 in the early night to degrade TIM. These results suggest that PDF neurons integrate light information and then modulate the phase of E cell oscillations and behavioral rhythms. The results also explain how fly brain rhythms persist in constant darkness and without CRY (Guo, 2014).

A PDF/NPF neuropeptide signaling circuitry of male Drosophila melanogaster controls rival-induced prolonged mating

A primary function of males for many species involves mating with females for reproduction. Drosophila melanogaster males respond to the presence of other males by prolonging mating duration to increase the chance of passing on their genes. To understand the basis of such complex behaviors, this study examined the genetic network and neural circuits that regulate rival-induced Longer-Mating-Duration (LMD). This study identified a small subset of clock neurons in the male brain that regulate LMD via neuropeptide signaling. LMD requires the function of pigment-dispersing factor (PDF) in four s-LN_v neurons and its receptor PDFR in two LNd neurons per hemisphere, as well as the function of neuropeptide F (NPF) in two neurons within the sexually dimorphic LNd region and its receptor NPFR1 in four s-LN_v neurons per hemisphere. Moreover, rival exposure modifies the neuronal activities of a subset of clock neurons involved in neuropeptide signaling for LMD (Kim, 2013).

This study provides evidence for the crucial involvement of two neuropeptides, PDF and NPF, in the modulation of reproductive behavior by the male's prior experience with other males. By identifying neurons required for this neuropeptide modulation, this study delineates the central neuronal circuitry and finds that the crucial neurons expressing a neuropeptide are not in synaptic contact with the crucial neurons expressing its receptor, providing further evidence for the long-range influence of neuropeptides. Remarkably, sharing housing with male rivals alters the activity of a subset of clock neurons, including those neurons expressing PDF and NPF that are crucial for this behavioral modulation. It was also found that these altered neuronal activities of PDF- and NPF-expressing neurons in group-reared males are dependent on the signaling by NPF and PDF, respectively (Kim, 2013).

LMD requires PDF expression in four s-LN_v neurons, and it also requires the expression of the NPF receptor, NPFR1, in those four s-LN_v neurons. These four s-LN_v neurons thus appear to act in the LMD generation as a relay station to receive NPF neuropeptide signaling and to transmit PDF neuropeptide signaling to neurons expressing the PDF receptor PDFR (Kim, 2013).

Unlike PDF-expressing neurons with well-known functions for circadian rhythm behavior, much less is known about neurons expressing PDFR. To search for the PDFR-expressing cells involved in LMD, a small number of CRY-positive, but PDF-negative, neurons required to generate LMD were identified. Various pdfR-GAL4 lines were used to identify LNd neurons and PI neurons as candidate PDFR-expressing neurons. After the involvement of PI neurons was ruled out, it was demonstrated that expressing PDFR in LNd neurons of pdfR mutants was sufficient to rescue the LMD deficits. Among this small group of CRY-positive, but PDF-negative, LNd neurons, two cells that express PDFR, but not NPF, and another distinct group of two sexually dimorphic cells that express NPF, but not PDFR, in each hemisphere are required for LMD. Moreover, the neuronal activities of these male-specific LNd neurons that express NPF were increased by the exposure to rivals, whereas the neuronal activity of PDF-expressing s-LN_v neurons appeared to be decreased by rival exposure. These four s-LN_v neurons also express NPFR1, which is coupled to Gi to mediate inhibition of adenylyl cylcase. Given that the rival exposure-induced alteration of s-LN_v neuronal activity requires NPFR1 function, one plausible scenario is that rival exposure increases the activity of NPF-expressing LNd neurons, which release NPF to activate NFPR1 on s-LN_v neurons so as to reduce the activity of these PDF-expressing s-LN_v neurons (Kim, 2013).

PDF appears to be released in a paracrine fashion to activate the G-protein-coupled receptor PDFR. PDFR is not found in the four s-LN_v neurons that express PDF. One LNd neuron is known to be PDFR-positive, though its PDFR signaling has not been characterized. The two PDFR-expressing LNd neurons per hemisphere were found to be crucial for LMD, and they do not form direct synaptic contact with the s-LN_v dorsal projections, consistent with the previous report that presynaptic terminals of PDF-expressing neurons have no direct contact with LNd neurons. Expression of the secreted form of PDF via an s-LN_v-specific GAL4 driver in pdf⁰¹ mutant could rescue the disrupted LMD; however, expression of a membrane-tethered form of PDF could not. In contrast, expression of a membrane-tethered form of PDF via pdfR(D)-GAL4(2), with restricted expression in LNd and PI neurons, could rescue the disrupted LMD phenotype of pdf⁰¹ mutants. These results indicate that PDF secreted from s-LN_v neurons can activate PDFR in LNd neurons to generate LMD. The dendrites of PDFR-positive LNd neurons labeled by pdfR(D)-GAL4(2) are located near the dorsal projections of PDF-expressing neurons. The dendrites of LNd neurons labeled by 50y-GAL4, which could impair LMD when it drives the expression of pdfR-siRNA to reduce PDFR activity in LNd neurons, also are located near these PDF-expressing neuronal projections. It has been reported that neuropeptide signaling does not require synaptic contacts. The released peptide may diffuse over tens of micrometers to reach its receptors, and the action of a peptide is limited by dilution as well as degradation/ inactivation by membrane-bound peptidases. Thus, PDF released from s-LN_v neuronal projections may signal nearby PDFR-positive LNd neurons via diffusion rather than direct synaptic contact. In summary, this study has identified two PDFR-positive LNd neurons per hemisphere that are responsible for generating LMD via PDF/PDFR signaling. It is suggested that PDF released from s-LN_v is responsible for PDFR signaling in these LNd neurons (Kim, 2013).

This study reveals that PDF and NPF signaling is crucial for the mating duration that is controlled by the male's experience with rivals. Moreover, rival exposure greatly reduced the activity of the s-LN_v neurons normalized by that of l-LN_v neurons both expressing PDF but increased the activity of LNd neurons normalized by that of D2 neurons both expressing NPF. Interestingly, this increase in neuronal activity of NPF-positive LNd neurons in group-rearing conditions is not observed in pdfR mutant animals. Given that PDFR and NPF are expressed by two distinct populations of LNd neurons, the requirement of PDFR function for the rival-induced modulation of NPF-expressing neuronal activity in the LNd region raises the intriguing question of whether neuronal signaling (perhaps involving another as-yet-unidentified neuropeptide) is involved in LMD (Kim, 2013).

A recent study has identified four abdominal ganglion (AG) interneurons (INs) that contain the neuropeptide corazonin (Crz) and modulate copulation duration. These neurons might play a role as a final set of effectors for the convergent effects of acute and chronic rival competition on the copulation duration. Elucidating the neural circuitry between these AG neurons and clock neurons would be helpful in furthering understanding of how male flies regulate mating duration in response to rivals (Kim, 2013).

Recent studies have shown that sexually dimorphic responses to pheromones in the nematode Caenorhabditis elegans may arise from differences in the balance of neural circuits during development or in the adult via neuromodulation. The current study adds to this emerging body of literature, illustrating the importance of sexually dimorphic neuromodulation via neuropeptide signaling in social behavior (Kim, 2013).

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_vs) are the dominant pacemaker neurons and set the pace of most other clock neurons in constant darkness. This study shows that two distinct G protein signaling pathways are required in LN_vs for 24 hr rhythms. Reducing signaling in LN_vs via the G alpha subunit Gs, which signals via cAMP, or via the G alpha subunit Go, which signals via Phospholipase 21c, lengthens the period of behavioral rhythms. In contrast, constitutive Gs or Go signaling makes most flies arrhythmic. Using dissociated LN_vs in culture, it was found that Go and the metabotropic GABA(B)-R3 receptor are required for the inhibitory effects of GABA on LN_vs 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_vs require GABAergic inputs for 24 hr rhythms (Dahdal, 2010).

The long-periods observed with reduced Gs signaling are consistent with four other manipulations of cAMP levels or PKA activity that alter fly circadian behavior. First, long-period rhythms with dnc over-expression complement the short periods of dnc hypomorphs and suggest that the latter are due to loss of dnc from LN_vs. dnc mutants also increase phase shifts to light in the early evening. However, this study found no difference in phase delays or advances between Pdf > dnc and control flies, suggesting that altered light-responses of dnc hypomorphs are due to dnc acting in other clock neurons. The period-altering effects seen when manipulating cAMP levels are also consistent with finding stat expressing the cAMP-binding domain of mammalian Epac1 in LN_vs lengthens period. This Epac1 domain likely reduces free cAMP levels in LN_vs, although presumably not as potently as UAS-dnc. Third, mutations in PKA catalytic or regulatory subunits that affect the whole fly disrupt circadian behavior. Fourth, over-expressing a PKA catalytic subunit in LN_vs rescues the period-altering effect of a UAS-shibire transgene that alters vesicle recycling, although the PKA catalytic subunit had no effect by itself. The long periods observed with reduced Gs signaling in LN_vs also parallel mammalian studies in which pharmacologically reducing Adenylate cyclase activity lengthened period in SCN explants and mice (Dahdal, 2010).

G-proteins typically transduce extracellular signals. What signals could activate Gs in s-LN_vs? PDF is one possibility since PDFR induces cAMP signaling in response to PDF in vitro, indicating that it likely couples to Gs. PDF could signal in an autocrine manner since PDFR is present in LN_vs. However, the long-periods observed with reduced Gs signaling differ from the short-period and arrhythmic phenotypes of Pdf and pdfr mutants. The likeliest explanation for these differences is that the altered behavior of Pdf and pdfr mutants results from effects of PDF signaling over the entire circadian circuit, whereas the current manipulations specifically targeted LN_vs. Indeed, LN_vs are not responsible for the short-period rhythms in Pdf⁰¹ null mutant flies. Other possible explanations for the differences between the long-period rhythms with decreased Gs signaling in LN_vs and the short-period rhythms of Pdf and pdfr mutants are that additional GPCRs couple to Gs in s-LN_vs and influence molecular clock speed and that the current manipulations decrease rather than abolish reception of PDF. In summary, the data shows that Gs signaling via cAMP in s-LN_vs modulates period length (Dahdal, 2010).

Go signaling via PLC21C constitutes a novel pathway that regulates the s-LN_v molecular clock. This study found that Go and the metabotropic GABA_B-R3 receptor are required for the inhibitory effects of GABA on larval LN_vs, which develop into adult s-LN_vs. The same genetic manipulations that block GABA inhibition of LN_vs in culture (expression of Ptx or GABA_B-R3-RNAi) lengthened the period of adult locomotor rhythms. Furthermore, the molecular clock in s-LN_vs is disrupted when a subset of GABAergic neurons are hyper-excited. Since the LN_vs do not produce GABA themselves, s-LN_vs require GABAergic inputs to generate 24hr rhythms. Thus s-LN_vs are less autonomous for determining period length in DD than previously anticipated (Dahdal, 2010).

Activation of G-proteins can have both short- and long-term effects on a cell. With Go signaling blocked by Ptx, short-term effects on LN_v responses were detected in response to excitatory ACh and longer-term effects on the molecular clock. The latter are presumably explained by PLC activation since the behavioral phenotypes of Pdf > GoGTP flies were rescued by reducing Plc21C expression (Dahdal, 2010).

Since s-LN_v clocks were unchanged even when the speed of all non-LN_v clock neurons were genetically manipulated, it is surprising to find s-LN_v clocks altered by signaling from GABAergic non-clock neurons. Why would LN_vs need inputs from non-clock neurons to generate 24hr rhythms? One possibility is that LN_vs receive multiple inputs which either accelerate or slow down the pace of their molecular clock but overall balance each other to achieve 24hr rhythms in DD. Since reducing signaling by Gs and Go lengthens period, these pathways normally accelerate the molecular clock. According to this model, there are unidentified inputs to LN_vs which delay the clock. Identifying additional receptors in LN_vs would allow this idea to be tested (Dahdal, 2010).

Previous work showed that GABAergic neurons project to LN_vs and that GABA_A receptors in l-LN_vs regulate sleep. The current data show that constitutive activation of Go signaling dramatically alters behavioral rhythms, suggesting that LN_vs normally receive rhythmic GABAergic inputs. But how can s-LN_vs integrate temporal information from non clock-containing GABAergic neurons? s-LN_vs could respond rhythmically to a constant GABAergic tone by controlling GABA_B-R3 activity. Indeed, a recent study found that GABA_B-R3 RNA levels in s-LN_vs are much higher at ZT12 than at ZT0 (Kula-Eversole, 2010). Strikingly, this rhythm in GABA_B-R3 expression is in antiphase to LN_v neuronal activity. Thus regulated perception of inhibitory GABAergic inputs could at least partly underlie rhythmic LN_v excitability. GABAergic inputs could also help synchronize LN_vs as in the cockroach circadian system. Thus GABA's short-term effects on LN_v excitability, likely mediated by Gβ/γ, and GABA's longer-term effects on the molecular clock via Go may both contribute to robust rhythms (Dahdal, 2010).

This work adds to the growing network view of circadian rhythms in Drosophila where LN_vs integrate information to set period for the rest of the clock network in DD. The period-altering effects of decreased G-protein signaling in LN_vs point to a less hierarchical and more distributed network than previously envisioned. Since the data strongly suggests that GABA inputs are novel regulators of 24hr rhythms, the GABAergic neurons that fine-tune the s-LN_v clock should be considered part of the circadian network (Dahdal, 2010).

PDF receptor expression reveals direct interactions between circadian oscillators in Drosophila

Daily rhythms of behavior are controlled by a circuit of circadian pacemaking neurons. In Drosophila, 150 pacemakers participate in this network, and recent observations suggest that the network is divisible into M and E oscillators, which normally interact and synchronize. Sixteen oscillator neurons (the small and large lateral neurons [LN_vs]) express a neuropeptide called pigment-dispersing factor (PDF) whose signaling is often equated with M oscillator output. Given the significance of PDF signaling to numerous aspects of behavioral and molecular rhythms, determining precisely where and how signaling via the PDF receptor (PDFR) occurs is now a central question in the field. This study shows that GAL4-mediated rescue of pdfr phenotypes using a UAS-PDFR transgene is insufficient to provide complete behavioral rescue. In contrast, an approximately 70-kB PDF receptor (pdfr) transgene is described that does rescue the entire pdfr circadian behavioral phenotype. The transgene is widely but heterogeneously expressed among pacemakers, and also among a limited number of non-pacemakers. These results support an important hypothesis: the small LN_v cells directly target a subset of the other crucial pacemaker neurons cells. Furthermore, expression of the transgene confirms an autocrine feedback signaling by PDF back to PDF-expressing cells. Finally, the results present an unexpected PDF receptor site: the large LN_v cells appear to target a population of non-neuronal cells that resides at the base of the eye (Im, 2010).

Functions of PDF receptor orthologs in other species

Pigment-dispersing factor (Pdf) signaling in the circadian system of Caenorhabditis elegans

The neuropeptide PDF (Pigment Dispersing Factor) is important for the generation and entrainment of circadian rhythms in the fruitfly Drosophila melanogaster. Recently two pdf homologs, pdf-1 and pdf-2, and a PDF receptor, pdfr-1, have been found in Caenorhabditis elegans and have been implicated in locomotor activity. This work studied the role of the PDF neuropeptide in the circadian system of C. elegans and found that both pdf-1 and pdf-2 mutants affect the normal locomotor activity outputs. In particular, loss of pdf-1 induced circadian arrhythmicity under both light-dark (LD) and constant dark (DD) conditions. These defects can be rescued by a genomic copy of the pdf-1 locus. These results indicate that PDF-1 is involved in rhythm generation and in the synchronization to LD cycles, since rhythmic patterns of activity rapidly disappear when pdf-1 mutants are recorded under both entrained and free running conditions. The role of PDF-2 and the PDF receptors is probably more complex and involves the interaction between the two pdf paralogues found in the nematode (Herrero, 2015).

Search PubMed for articles about Drosophila PDF receptor

Collins, B., Kaplan, H. S., Cavey, M., Lelito, K. R., Bahle, A. H., Zhu, Z., Macara, A. M., Roman, G., Shafer, O. T. and Blau, J. (2014). Differentially timed extracellular signals synchronize pacemaker neuron clocks. PLoS Biol 12(9): e1001959. PubMed ID: 25268747

Dahdal, D., Reeves, D. C., Ruben, M., Akabas, M. H. and Blau, J. (2010). Drosophila pacemaker neurons require g protein signaling and GABAergic inputs to generate twenty-four hour behavioral rhythms. Neuron 68(5): 964-977. PubMed ID: 21145008

Duvall, L. B. and Taghert, P. H. (2012). The circadian neuropeptide PDF signals preferentially through a specific adenylate cyclase isoform AC3 in M pacemakers of Drosophila. PLoS Biol 10(6): e1001337. PubMed ID: 22679392

Duvall, L. B. and Taghert, P. H. (2013). E and M circadian pacemaker neurons use different PDF receptor signalosome components in drosophila. J Biol Rhythms 28(4): 239-248. PubMed ID: 23929551

Goda, T., Tang, X., Umezaki, Y., Chu, M. L., Kunst, M., Nitabach, M. N. N. and Hamada, F. N. (2016). Drosophila DH31 neuropeptide and PDF receptor regulate night-onset temperature preference. J Neurosci 36(46): 11739-11754. PubMed ID: 27852781

Goda, T., Umezaki, Y., Alwattari, F., Seo, H. W. and Hamada, F. N. (2019). Neuropeptides PDF and DH31 hierarchically regulate free-running rhythmicity in Drosophila circadian locomotor activity. Sci Rep 9(1): 838. PubMed ID: 30696873

Gorska-Andrzejak, J., Chwastek, E. M., Walkowicz, L. and Witek, K. (2018). On variations in the level of PER in glial clocks of Drosophila optic lobe and its negative regulation by PDF signaling. Front Physiol 9: 230. PubMed ID: 29615925

Guo, F., Cerullo, I., Chen, X. and Rosbash, M. (2014). PDF neuron firing phase-shifts key circadian activity neurons in Drosophila. Elife 3. PubMed ID: 24939987

Guo, F., Yu, J., Jung, H. J., Abruzzi, K. C., Luo, W., Griffith, L. C. and Rosbash, M. (2016). Circadian neuron feedback controls the Drosophila sleep--activity profile. Nature 536(7616): 292-297. PubMed ID: 27479324

Hajji, K., Mteyrek, A., Sun, J., Cassar, M., Mezghani, S., Leprince, J., Vaudry, D., Masmoudi-Kouki, O. and Birman, S. (2019). Neuroprotective effects of PACAP against paraquat-induced oxidative stress in the Drosophila central nervous system. Hum Mol Genet. PubMed ID: 30715303

Head, L.M., et al. (2015). The influence of light on temperature preference in Drosophila. Curr Biol 25(8):1063-8. PubMed ID: 25866391

Herrero, A., Romanowski, A., Meelkop, E., Caldart, C., Schoofs, L. and Golombek, D. A. (2015). Pigment-dispersing factor (Pdf) signaling in the circadian system of Caenorhabditis elegans. Genes Brain Behav. PubMed ID: 26113231

Im, S. H. and Taghert, P. H. (2010). PDF receptor expression reveals direct interactions between circadian oscillators in Drosophila. J Comp Neurol 518(11): 1925-1945. PubMed ID: 20394051

Kaneko, H., Head, L. M., Ling, J., Tang, X., Liu, Y., Hardin, P. E., Emery, P. and Hamada, F. N. (2012). Circadian rhythm of temperature preference and its neural control in Drosophila. Curr Biol 22(19): 1851-1857. PubMed ID: 22981774

Kim, W. J., Jan, L. Y. and Jan, Y. N. (2013). A PDF/NPF neuropeptide signaling circuitry of male Drosophila melanogaster controls rival-induced prolonged mating. Neuron 80: 1190-1205. PubMed ID: 24314729

Kunst, M., Hughes, M. E., Raccuglia, D., Felix, M., Li, M., Barnett, G., Duah, J. and Nitabach, M. N. (2014). Calcitonin gene-related peptide neurons mediate sleep-specific circadian output in Drosophila. Curr Biol 24(22): 2652-2664. PubMed ID: 25455031

Langlet, C,, Langer, I., Vertongen, P., Gaspard, N., Vanderwinden, J. M. and Robberecht, P. (2005). Contribution of the carboxyl terminus of the VPAC1 receptor to agonist-induced receptor phosphorylation, internalization, and recycling. J Biol Chem. 280: 28034-28043. PubMed ID: 15932876

Li, W., Trigg, J. S. and Taghert, P. H. (2022). Regulation of PDF receptor signaling controlling daily locomotor rhythms in Drosophila. PLoS Genet 18(5): e1010013. PubMed ID: 35605015

Li, Y., Guo, F., Shen, J. and Rosbash, M. (2014). PDF and cAMP enhance PER stability in Drosophila clock neurons. Proc Natl Acad Sci U S A 111(13): E1284-1290. PubMed ID: 24707054

Liu, S., Lamaze, A., Liu, Q., Tabuchi, M., Yang, Y., Fowler, M., Bharadwaj, R., Zhang, J., Bedont, J., Blackshaw, S., Lloyd, T. E., Montell, C., Sehgal, A., Koh, K. and Wu, M. N. (2014). WIDE AWAKE mediates the circadian timing of sleep onset. Neuron 82(1): 151-166. PubMed ID: 24631345

Potdar, S. and Sheeba, V. (2018). Wakefulness is promoted during day time by PDFR signalling to dopaminergic neurons in Drosophila melanogaster. eNeuro 5(4). PubMed ID: 30131970

Seluzicki, A., Flourakis, M., Kula-Eversole, E., Zhang, L., Kilman, V. and Allada, R. (2014). Dual PDF Signaling Pathways Reset Clocks Via TIMELESS and Acutely Excite Target Neurons to Control Circadian Behavior. PLoS Biol 12: e1001810. PubMed ID: 24643294

Song, Q., Feng, G., Zhang, J., Xia, X., Ji, M., Lv, L. and Ping, Y. (2017). NMDA receptor-mediated Ca2+ influx in the absence of Mg2+ block disrupts rest:activity rhythms in Drosophila. Sleep 40(12). PubMed ID: 29029290

Vecsey, C. G., Pirez, N. and Griffith, L. C. (2014). The Drosophila neuropeptides PDF and sNPF have opposing electrophysiological and molecular effects on central neurons. J Neurophysiol 111(5): 1033-1045. PubMed ID: 24353297

date revised: 4 April 2022

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