InteractiveFly: GeneBrief

Pigment-dispersing factor receptor: Biological Overview | References

Gene Name - Pigment-dispersing factor receptor

Synonyms -

Cytological map position - 3A4-3A6

Function - G-protein coupled receptor

Keywords - dopaminergic neurons that respond to PDFR signalling are sleep-promoting and during the day when PDF levels are high, they are inhibited, thereby promoting wakefulness, regulates free-running rhythmicity in Drosophila circadian locomotor activity, modulators of molecular cycling in the peripheral clocks of both the glial cells and the photoreceptors of the compound eye, regulation of night-onset temperature preference

Symbol - Pdfr

FlyBase ID: FBgn0260753

Genetic map position - chrX:2,552,206-2,578,640

NCBI classification - 7tm_GPCRs: seven-transmembrane G protein-coupled receptor superfamily

Cellular location - surface transmembrane

NCBI links: EntrezGene, Nucleotide, Protein

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-LNv) 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 LNv (sLNv) 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 (LNvs) 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 LNv (l-LNv) 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 LNv; s-LNv) 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-LNv 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-LNv 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-LNv promotes the inhibition of sleep-promoting dopaminergic neurons by PDFR signaling. The identity of dopamine neurons acting on l-LNv and those responding to PDFR signaling may differ which can be uncovered with additional experiments (Potdar, 2018).

The role of s-LNv in modulating sleep and wake has been explored in some detail in the recent years. s-LNv have also been shown to promote sleep via short NPF (sNPF) as well as myoinhibitory peptide (MiP) by inhibiting the wake-promoting l-LNv. This study shows that PDF+ s-LNv 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-LNv in modulating wake-promoting effects of l-LNv. Yet, how this wake-promoting signal which originates in the l-LNv gets relayed to the s-LNv is not understood. Furthermore, from the screen it is clear that this function is not mediated via PDFR signaling among the LNv, as downregulating and overexpressing pdfr in s-LNv (Clk 9M GAL4 and Pdf GAL4) do not result in any sleep defects. Thus, l-LNv to s-LNv wake-promoting signal is independent of PDF while s-LNv 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-LNv, 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 LNv 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-LNv) 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-LNv. 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 LNv 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, LNvs, and acts on other clock cells through PDFR to synchronize and maintain robust molecular rhythms. PDF expression from LNvs in Dh31#51;Pdf01 mutants restored rhythmicity, in contrast to tethered-PDF (t-PDF) expression in LNvs, indicating that an autoreceptor of PDF signals in LNvs 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 sLNvs28. 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 LNvs 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 LNvs was not detectable via anti-DH31 antibody staining, a recent RNA-seq analysis detected Dh31 gene expression in both LNvs and DN1s. Therefore, DH31 expression from LNvs 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 sLNvs 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 Dh31r1/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;Dh31r1/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 Dh31r1/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, Dh31r1/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 LNvs 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).

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 (LNvs) 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 LNvs 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 LNv 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 LNv 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-LNvs), large ventral LNs (l-LNvs), 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 perS ameliorates the phenotypes of pdf-null flies. The period protein (Per) is a well-studied repressor of clock gene transcription, and the perS 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 perS gene therefore suggests that perS 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 perS 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 LNvs, 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-LNvs 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-LNvs as well as the s-LNvs, 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-LNv dorsal projections. In addition, the l-LNvs 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-LNvs 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 perS and the perS;;pdf01 strains. A residual period-lengthening effect of PDF suggests that perS does not endow all oscillators with the identical period, i.e., that there is still some asynchrony between different perS 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 perS 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 perS-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 (LNvs) or two dorsal clock neurons (DN1s). Unexpectedly, it was found that the PDF Receptor (PdfR) is required in both LNvs and DN1s to maintain synchronized LNv clocks. It was also found that glutamate is a second synchronizing signal that is released from DN1s and perceived in LNvs via the metabotropic glutamate receptor (mGluRA). Because simultaneously reducing Pdfr and mGluRA expression in LNvs severely dampened Timeless clock protein oscillations, it is concluded that the master pacemaker LNvs require extracellular signals to function normally. These two synchronizing signals are released at opposite times of day and drive cAMP oscillations in LNvs. Finally it was found that PdfR and mGluRA also help synchronize Timeless oscillations in adult s-LNvs. 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-LNv 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-LNv 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-LNv neurons, and it also requires the expression of the NPF receptor, NPFR1, in those four s-LNv neurons. These four s-LNv 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-LNv neurons appeared to be decreased by rival exposure. These four s-LNv neurons also express NPFR1, which is coupled to Gi to mediate inhibition of adenylyl cylcase. Given that the rival exposure-induced alteration of s-LNv 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-LNv neurons so as to reduce the activity of these PDF-expressing s-LNv 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-LNv 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-LNv 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-LNv-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-LNv 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-LNv 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-LNv 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-LNv neurons normalized by that of l-LNv 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-LNvs) 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 LNvs for 24 hr rhythms. Reducing signaling in LNvs 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 LNvs in culture, it was found that Go and the metabotropic GABA(B)-R3 receptor are required for the inhibitory effects of GABA on LNvs 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-LNv molecular clocks. Therefore, s-LNvs 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 LNvs. 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 LNvs lengthens period. This Epac1 domain likely reduces free cAMP levels in LNvs, 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 LNvs 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 LNvs 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-LNvs? 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 LNvs. 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 LNvs. Indeed, LNvs 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 LNvs and the short-period rhythms of Pdf and pdfr mutants are that additional GPCRs couple to Gs in s-LNvs 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-LNvs modulates period length (Dahdal, 2010).

Go signaling via PLC21C constitutes a novel pathway that regulates the s-LNv molecular clock. This study found that Go and the metabotropic GABAB-R3 receptor are required for the inhibitory effects of GABA on larval LNvs, which develop into adult s-LNvs. The same genetic manipulations that block GABA inhibition of LNvs in culture (expression of Ptx or GABAB-R3-RNAi) lengthened the period of adult locomotor rhythms. Furthermore, the molecular clock in s-LNvs is disrupted when a subset of GABAergic neurons are hyper-excited. Since the LNvs do not produce GABA themselves, s-LNvs require GABAergic inputs to generate 24hr rhythms. Thus s-LNvs 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 LNv 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-LNv clocks were unchanged even when the speed of all non-LNv clock neurons were genetically manipulated, it is surprising to find s-LNv clocks altered by signaling from GABAergic non-clock neurons. Why would LNvs need inputs from non-clock neurons to generate 24hr rhythms? One possibility is that LNvs 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 LNvs which delay the clock. Identifying additional receptors in LNvs would allow this idea to be tested (Dahdal, 2010).

Previous work showed that GABAergic neurons project to LNvs and that GABAA receptors in l-LNvs regulate sleep. The current data show that constitutive activation of Go signaling dramatically alters behavioral rhythms, suggesting that LNvs normally receive rhythmic GABAergic inputs. But how can s-LNvs integrate temporal information from non clock-containing GABAergic neurons? s-LNvs could respond rhythmically to a constant GABAergic tone by controlling GABAB-R3 activity. Indeed, a recent study found that GABAB-R3 RNA levels in s-LNvs are much higher at ZT12 than at ZT0 (Kula-Eversole, 2010). Strikingly, this rhythm in GABAB-R3 expression is in antiphase to LNv neuronal activity. Thus regulated perception of inhibitory GABAergic inputs could at least partly underlie rhythmic LNv excitability. GABAergic inputs could also help synchronize LNvs as in the cockroach circadian system. Thus GABA's short-term effects on LNv 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 LNvs integrate information to set period for the rest of the clock network in DD. The period-altering effects of decreased G-protein signaling in LNvs 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-LNv 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 [LNvs]) 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 LNv 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 LNv 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

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Biological Overview

date revised: 10 November 2019

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