InteractiveFly: GeneBrief

Pigment-dispersing factor: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

Pigment-dispersing hormones (PDH) are neuropeptides secreted from the sinus gland of the eyestalks of crustaceans. Their physiological functions in crustaceans include translocation of retinal distal pigments and epithelial chromatophoral pigment dispersion (Rao, 1993). All PDHs identified so far are octadecapeptides, and their C-terminals are invariably modified by alpha-amidation. Because their physiological roles have not been clearly defined in insects, they are known as pigment-dispersing factors (PDFs). The insect peptides have substantial pigment-dispersing activies when assayed on the eyestalkless fiddler crab. However, PDFs are unlikely to play a role in cuticular pigment migration, since insects do not possess the epithelium chromatophore found in crustaceans (Park, 1998 and references therein).

Cloning the Drosophila Pdf gene is a first step toward unraveling its biological function. The Pdf gene has been cloned using RACE (rapid amplification of cDNA ends). The design of the degenerate primer used for cloning was based on the amino acid sequence homology among members of the PDH family. Adult heads were chosen as the source of RNA, since immunocytochemical studies have shown more PDH-immunoreactive cells present in the adult Drosophila brain than in the abdominal ganglion (Park, 1998).

Several lines of evidence suggest that PDFs might play a role in the regulation of insect biological rhythms. Immunocytochemical studies, using antibodies against crustacean PDHs, reveal three groups of PDH-immunoreactive neurons in the brain of orthopteroid insects. One group of cells, known as PDFMe for its location at the anterior edge of the medulla, fulfills the anatomical criteria proposed for the cardiac pacemakers in these insects. Lesion studies, along with immunocytochemistry and behavioral analysis in the cockroach, suggest that PDFMe might be part of the circadian pacemaker in this insect (Stengl, 1994). Cell bodies of Drosophila PDH-immunoreactive neurons are located at the anterior base of the medulla; their axonal arborizations are well suited to modulate the neuronal activities in the optic lobe and relay the circadian information to the midbrain (Helfrich-Foster, 1995 and 1997). In adults, this group consists of four to six neurons with large somata (large PDFMe neurons) and four neurons with small somata (small PDFMe neurons). Both the small and the large PDFMe neurons are identical to the ventral lateral neurons, a group of neurons containing the Period protein (Helfrich-Forster, 1997). beta-PDH-like antigens colocalize with those detected by a new antibody to distinct sequences of the Drosophila proPDF precursor (anti-PAP). Pdf gene products are found in three cell types. These include the LN_vs, two to four tritocerebral cells (PDF-Tri), and four to six abdominal cells (PDF-Ab) (Renn 1999).

An evaluation was made of the activity rhythms of flies with PDF cell ablations. Ablation was accomplished by ectopic expression of the cell death genes reaper or head involution defective in cells that normally express Pdf. No PDF-positive cell bodies are detected in the CNS of third instar larvae bearing pdf-GAL4 and UAS-rpr. In some of these larval CNSs, however, a few residual processes in the dorsal brain are very faintly stained. In adults bearing both transgenes, neither s-LN_v cell bodies nor dorsal processes are stained, whereas some l-LN_vs are stained. p35 encodes a caspase inhibitor that can rescue rpr- or hid-mediated cell death. When p35 is coexpressed with rpr, most of the larval LNs (80%), adult s-LN_vs (70%), and l-LN_vs (80%) survive. The PDF-Tri neurons (which normally cease to express pdf after adult days 1-2) remain persistently PDF immunoreactive in rpr-rescued brains, up to adult day 10. This suggests that PDF-Tri cells normally die in young adults (Renn, 1999).

Analysis of DD behavior shows that 63% of rpr-ablated flies are rhythmic for the entire period, whereas only 17% of hid-ablated flies sustain such rhythmicity. As in the case of pdf⁰¹, separate periodogram analysis of DD days 3-9 reveals decreased proportions of rhythmic individuals in cell-ablated flies. The subnormal SNR values computed for both the rpr- and hid-ablated flies are consistent with their abnormal free-running behavior. Finally, the rpr and hid ablation individuals that are persistently rhythmic in DD tend to manifest short circadian periods (Renn, 1999).

Despite having a slightly lower number of PDF cells, animals that coexpressed rpr and p35 display essentially normal behavior. The free-running period of the rescued group is approximately 0.5 hr longer than control values. In contrast to animals that coexpress p35 and rpr, only about 70% animals that coexpress p35 and hid are rhythmic in DD, and the SNRs for this group are intermediate between those for controls and for hid ablation. This incomplete behavioral rescue parallel the histological findings (Renn, 1999).

What then is the biological function, if any, of Pdf in the photoperiod response? Isolation of mutations in the Pdf gene has allowed this question to be addressed. The most severe phenotype displayed by pdf⁰¹ mutants and by PDF cell-ablated animals is that the majority are arrhythmic in constant darkness (DD). Both sets of animals are rhythmic over the first 1-2 days of constant darkness. Their locomotor patterns become arrhythmic gradually over a 9 day period. It is concluded that circadian behavior is largely independent of Pdf hormone and of LN_v neurons during normal light dark (LD) cycles and short term continuous darkness, but the requirements for Pdf and the cells that produce the hormone are revealed during artificially sustained constant DD conditions (Renn, 1999).

What features of LN_v neurons and pdf signaling could explain this phenotype? In cockroaches, injection of beta-PDH into the brain causes phase delays in daily locomotor activity, consistent with a role for the peptide in a nonphotic clock input (Petri, 1997). The morphology of l-LN_v neurons suggests a basis for how secreted Pdf gene products could access the pacemaker neurons (Kaneko, 1997). A subset of l-LN_v cells projects axons across the midline to the area containing the contralateral LN_v cell bodies. Therefore, rhythmic l-LN_v release of PDF could produce a phase delay in pacemakers of the opposite side and, thus, contribute to bilateral synchrony (Renn, 1999).

This scenario predicts that in Drosophila mutant for Pdf, the circadian clock will operate with advanced phase in LD and run more quickly in DD. These behaviors are the same as those observed for Pdf-null animals. The deterioration of free-running rhythmicity over DD days 1-3 may therefore reflect a gradual loss of synchronization between bilateral pacemaker centers. The disconnected mutant displays a progressive damping of rhythmicity and also lacks LN_v neurons (Helfrich-Forster, 1998). Likewise, the ablation of the avian pineal gland produces an analogous behavior in sparrows: when transferred from LD cycling to DD conditions, operated animals display a progressive loss of behavioral rhythmicity (Menaker, 1976). This effect has been shown to derive from lack of melatonin, which normally helps to maintain a mutual synchronization between the pineal and other pacemaker structures (Cassone, 1984).

The dorsally projecting s-LN_v cells may have a greater role in regulating circadian locomotor rhythms than the l-LN_vs. Cell ablation studies are consistent with the proposition that a single LN_v is competent to organize behavioral rhythmicity. This same conclusion was reached in an analysis of disco mutants (Helfrich-Forster, 1998). Likewise, the circadian regulators Clock and cycle regulate pdf expression in s-LN_vs, but not in l-LN_vs (J. H. Park. and C. Helfrich-Förster, et al., unpublished data cited in Renn, 1999). That result argues that neuropeptide expression by s-LNs neurons is especially important for the circadian behavioral regulation that has been inferred from analysis of pdf⁰¹ animals (Renn, 1999).

While the current results strongly support the hypothesis that LN_vs are critical circadian pacemaker neurons, analyses of both pdf⁰¹ and PDF-ablated flies reveal minorities of animals that maintain weak rhythmicity in DD. These low proportions of rhythmic individuals suggest the involvement of secondary pacemaker neurons involved in the circadian regulation of behavior. Their cellular identities are unknown, but on the assumption that they will express the clock genes period and timeless, three specific candidates are proposed. The first is a fifth per-positive, pdf-negative LN_v neuron; a cell with such properties was found in larvae, and it may also exist in adults; if so, its activities are presumed not to be affected in Pdf mutants. The second candidate cell type is represented by the dorsolateral LN_d cluster of neurons. The third plausible candidate cell type is represented by the Dorsal Neurons (DNs) of posterior-medial brain regions. The second and third candidate cell types are both Pdf negative (Helfrich-Forster, 1995). Free-running rhythms are more severely disrupted in disco flies than in pdf⁰¹ flies, and it is proposed that this is because disco flies lack almost all per-positive LN neurons, not just the ventral LN group (Renn, 1999 and references therein).

The pdf⁰¹ and neuron-ablated animals entrain to a 24 hr light:dark cycle and show considerable rhythmicity. This feature is noticeably different from other clock mutants, which are solely driven by photoperiod. This difference suggests the clock is still running in pdf⁰¹ and cell-ablated flies and that PDF is therefore not a central component of the clock mechanism. However, both pdf⁰¹ and cell-ablated flies display phase-advanced evening activity peaks in LD, and if rhythmic in DD, they display a short free-running period. It is proposed that the same physiological mechanism underlies both of these phenotypes. Both features are also displayed by per^Clk and norpA mutants. Light resets these fast-paced clocks by about 1 hr per day to 24 hr. Hence, pdf⁰¹, like per^Clk and norpA mutants, produces fast-paced clock movements in LD and DD. However, pdf⁰¹ variants have additional phenotypes: they fail to anticipate a lights-on transition in LD and are largely arrhythmic in DD. This suggests that a fast clock is not the only or appropriate explanation for all phenotypes associated with the pdf⁰¹-mutated and cell-ablated flies (Renn, 1999 and references therein).

The predicted Pdf gene product is a neuropeptide precursor, pro-PDF, which is presumed to be processed to two or more final peptide products that include the PAP and amidated 18-amino acid PDF molecules (Nassel, 1993). The pharmacological activities of injected beta-PDH peptide in other insects (Pyza, 1996; Petri, 1997) are consistent with the hypothesis that it represents a secreted agent. While the results of this study suggest that PDF is the principal circadian messenger in Drosophila, certain details remain ambiguous and will require further study. Two sets of results warrant comment. (1) A role for PDF neurons does did not implicate LN_v neurons exclusively. The lack of transmitter in PDF-Tri and PDF-Ab neurons, or their genetic ablation, may have contributed to the phenotypic defects. This is considered unlikely, as neither cell type expresses clock genes. Furthermore, PDF-Tri cells normally undergo apoptosis before the stage when locomotion is measured. (2) The experiments described here do not tell when the lesions studied have their effects: lack of transmitter or lack of Pdf neurons at an early, preadult stage may covertly affect behavioral periodicity, as well as having later physiological effects. The normal morphology of LN_v neurons in mutant animals argues against this possibility, and PDF neuropeptides have not previously been implicated in developmental functions. However, future experiments employing conditional manipulations will be necessary to evaluate this possibility (Renn, 1999 and references therein).

The role of Pdf in the Drosophila circadian system is notable, because it is a neuropeptide essential for circadian rhythm output. In rodents, the vasopressin neuropeptide gene is rhythmically expressed in SCN under the influence of the Clock gene (Jin, 1999). However, a functional role for vasopressin in SCN regulation of locomotor behavior has not been defined. Several neuropeptides can reset the phase of daily rhythms, but their effects appear to mimic natural inputs to circadian cycling, not its output. Norepinephrine is released from sympathetic nerve terminals in a circadian manner, but these neurons represent a distant, polysynaptic target of the neuronal output emanating from SCN. To date, PDF-related peptides have been found only in arthropods and mollusks. Whether related PDF-like peptides have analogous circadian functions in vertebrates, or whether a nonrelated transmitter has such functions, remains to be determined (Renn, 1999 and references therein).

The circadian neuropeptide PDF signals preferentially through a specific adenylate cyclase isoform AC3 in M pacemakers of Drosophila

The neuropeptide Pigment Dispersing Factor (PDF) is essential for normal circadian function in Drosophila. It synchronizes the phases of M pacemakers, while in E pacemakers it decelerates their cycling and supports their amplitude. The PDF receptor (PDF-R) is present in both M and subsets of E cells. Activation of PDF-R stimulates cAMP increases in vitro and in M cells in vivo. The present study asks: What is the identity of downstream signaling components that are associated with PDF receptor in specific circadian pacemaker neurons? Using live imaging of intact fly brains and transgenic RNAi, this study shows that adenylate cyclase AC3 underlies PDF signaling in M cells. Genetic disruptions of AC3 specifically disrupt PDF responses: they do not affect other Gs-coupled GPCR signaling in M cells, they can be rescued, and they do not represent developmental alterations. Knockdown of the Drosophila AKAP-like scaffolding protein Nervy also reduces PDF responses. Flies with AC3 alterations show behavioral syndromes consistent with known roles of M pacemakers as mediated by PDF. Surprisingly, disruption of AC3 does not alter PDF responses in E cells -- the PDF-R(+) LNd. Within M pacemakers, PDF-R couples preferentially to a single AC, but PDF-R association with a different AC(s) is needed to explain PDF signaling in the E pacemakers. Thus critical pathways of circadian synchronization are mediated by highly specific second messenger components. These findings support a hypothesis that PDF signaling components within target cells are sequestered into 'circadian signalosomes,' whose compositions differ between E and M pacemaker cell types (Duvall, 2012).

Loss of the PDF peptide or its receptor leads to abnormalities in circadian locomotor behavior, including a reduction in morning anticipatory peak and a phase advance of the evening anticipatory peak under 12:12 LD. Under constant conditions these flies show high levels of arrhythmicity or short, weak rhythms. PDF controls the amplitude and phase of molecular rhythms of pacemaker cells (Duvall, 2012).

PDF's role in synchronization of clock cells indicates that its mechanism of action is largely within the cells of the clock network. The PDF neuropeptide is expressed by two specific pacemaker subgroups (large and small LNvs) and the PDF receptor is expressed widely, although not uniformly, throughout the circadian network in both M and E cell groups (Im, 2010). The PDF receptor signals through calcium and cAMP, although specific signaling components remain unknown. Signaling can be demonstrated in nearly all pacemaker cell groups in vivo. Previous work indicates that M cells increase cAMP levels in response to at least two neuropeptides, PDF and DH31 (Shafer, 2008). The PDF and DH31 receptors belong to the same class II (secretin) G-protein coupled receptor (GPCR) family. Both PDF and DH31 receptors stimulate adenylate cyclases (AC) to produce cAMP in vitro, and in M cells in vivo (Shafer, 2008), but the specific downstream components that differentiate the two peptide receptors remain unknown. Likewise, the basis for PDF's differential actions on the molecular oscillator in different pacemakers has not yet been explained (Duvall, 2012).

The present study asks: What is the identity of downstream components that are associated with PDF-R signaling pathways in different circadian pacemaker neurons? Specifically, using live imaging of intact fly brains, the particular adenylate cyclase (AC) isoform was identified that is associated with PDF signaling in small LNv-commonly called M cells. Although some signaling components are common to both DH31 and PDF neuropeptide signaling, DH31 signaling does not require the same AC in the small LNv cells. This suggests that PDF signals preferentially through its favored AC, while other GPCRs, in the same identified pacemaker neurons, couple to other ACs. In addition, AC3 manipulations have no effect on PDF-R expressing LNd cells, part of the E cell network. Thus in Drosophila, critical pathways of circadian synchronization are mediated by at least two, highly specific second messenger pathways (Duvall, 2012).

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).

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 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 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).

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 Dh31r^1/Df mutants and flies undergoing Dh31r knockdown in their neurons showed normal rhythmicity in the locomotor activity rhythm25. 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 phenotype25, 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 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).

Light-mediated circuit switching in the Drosophila neuronal clock network

The circadian clock is a timekeeper but also helps adapt physiology to the outside world. This is because an essential feature of clocks is their ability to adjust (entrain) to the environment, with light being the most important signal. Whereas cryptochrome-mediated entrainment is well understood in Drosophila, integration of light information via the visual system lacks a neuronal or molecular mechanism. This study shows that a single photoreceptor subtype is essential for long-day adaptation. These cells activate key circadian neurons, namely the large ventral-lateral neurons (lLNvs), which release the neuropeptide pigment-dispersing factor (PDF). RNAi and rescue experiments show that PDF from these cells is necessary and sufficient for delaying the timing of the evening (E) activity in long-day conditions. This contrasts to PDF that derives from the small ventral-lateral neurons (sLNvs), which are essential for constant darkness (DD) rhythmicity. Using a cell-specific CRISPR/Cas9 assay, this study shows that lLNv-derived PDF directly interacts with neurons important for E activity timing. Interestingly, this pathway is specific for long-day adaptation and appears to be dispensable in equinox or DD conditions. The results therefore indicate that external cues cause a rearrangement of neuronal hierarchy, which contributes to behavioral plasticity (Schlichting, 2019).

Circadian clocks evolved as an adaptation to the continuous change of day and night and are believed to provide organisms a fitness advantage. The underlying molecular machinery includes a transcriptional-translational feedback loop, which generates oscillations of clock gene expression with an endogenous period close to 24 h. This period is approximately 24.2 h in humans, whereas a Drosophila period was reported to be 23.8 h. A key feature of circadian clocks is the ability to entrain to the 24 h environment. This means that the human clock has to be accelerated by about 0.2 h each day, whereas this Drosophila clock has to be slowed down to the same extent. To do so, clocks must integrate external cues, so-called zeitgebers, which are used to synchronize the molecular and physiological properties of the organism (Schlichting, 2019).

The most important zeitgeber is light. In mammals, a combination of the traditional photoreception pathway (rods and cones) and the circadian photoreceptor melanopsin in retinal ganglion cells allows for fine-tuning of clock synchronization. Similarly, Drosophila uses the visual system and possibly Rhodopsin 7 (Rh7) within the clock neurons as well as the circadian photoreceptor cryptochrome (CRY) for light synchronization. CRY-mediated entrainment is well understood in Drosophila, whereas less is known about the mechanism of entrainment via the visual system. It consists of seven eye structures: three ocelli, two Hofbauer-Buchner eyelets, and two compound eyes (Schlichting, 2019).

The compound eye consists of approximately 800 ommatidia, each harboring 8 photoreceptor cells (Rs): R1-6 are located in the periphery and span the whole depth of the ommatidium. These cells were previously shown to be important for motion vision and express Rhodopsin 1 (Rh1). In the center, R7 is located above R8. These cells have a complex expression pattern of Rh4 and/or Rh3 in R7 and Rh5 or Rh6 in R8. Although the mechanism of light transduction from the visual system to the central clock is still not completely understood, recent work indicates a special role for R8. These cells specifically target the sLNvs in standard conditions of 12 h light and 12 h darkness (LD 12:12). R8 photoreceptors additionally express and react to HisCl1 and can therefore not only act as photoreceptors but also as interneurons (Alejevski, 2109; Schlichting, 2019 and references therein).

Recent electrophysiological results further suggest that the visual system is able to activate an array of circadian clock neurons, e.g., it can activate the small ventral-lateral neurons (sLNvs), an important center for morning (M) activity. Furthermore, the visual system increases neuronal firing in the large LNvs (lLNvs), the arousal center within the circadian network. The 5th sLNv and the NPF+ dorsal-lateral neurons (LNds), previously implicated as necessary for evening (E) activity, also increase their firing rates in response to visual system stimulation. In addition, the visual system activates several dorsal neurons (DNs), which were recently implicated in connecting the circadian clock to central brain sleep centers. These data suggest that visual input is integrated into the clock network in a parallel fashion, which contradicts a master-oscillator point of view. The latter posits that these are the pigment-dispersing factor (PDF)-expressing neurons (sLNvs and lLNvs), which receive light input and release PDF upon illumination, thereby adjusting their downstream target neurons to the LD cycle (Schlichting, 2019).

To investigate the impact of the visual input pathway at the behavioral and neuronal level, this study investigated fly behavior under long-day conditions. Long days cause plastic changes in fly behavior: in standard light-dark cycles (LD 12:12), flies show a bimodal activity pattern with a M anticipation peak around lights on and an E anticipation peak around lights off; this results in a phase relationship of approximately 12 h between the two peaks appropriately adjust their peak timings, i.e., their E peak is phase advanced compared to wild-type flies. Moreover, visual input appears to modulate PDF release from the lateral neurons, which in turn modulates cells important for E activity. In summary, complex interactions between CRY- and PDF-expressing neurons appear to be essential for the behavior under long days: expressing these proteins in different parts of the fly brain alters the behavior of flies and mimics the behavior of high-latitude species using Drosophila melanogaster as a model. It is still unknown, however, which receptors and which neuronal pathways are involved in this adjustment (Schlichting, 2019).

This study shows that R8 of the compound eyes is essential for long-day adaptation. These photoreceptor cells connect to the PDF-containing lLNvs and trigger the release of this neuropeptide. Using a cell-specific CRISPR/Cas9 strategy, it was demonstrated that light-mediated PDF directly signals to the PDF receptor (PDFR) on E cells and hence delays E activity. The data implicate a mammal-like structure of clock entrainment, with the visual system activating PDF-expressing clock neurons. These data further support a shift of PDF targets between LD and constant darkness (DD) conditions as well as a more quantitative reorganization of neuronal dominance within the clock network by changes in photoperiod (Schlichting, 2019).

The circadian clock is able to entrain to the changes of day and night, with light being the most important zeitgeber. The adaptation to summer-like days is especially important for insects, as they are prone to predator visibility and even more importantly desiccation. Therefore, the circadian clock has to be plastic and be able to adjust behavior in response to changing environments. For example, flies show an additional afternoon peak during summer days under semi-natural conditions, which is thought to be an escape response of the fly from heat. This study shows that Drosophila adjusts its behavior to extremely long photoperiods (LD 20:4) by delaying its E peak as reported previously. Even though this photoperiod can only be found in very northern countries, Drosophila melanogaster is still able to adjust to this extreme light condition, which exemplifies its ability to adapt to various environmental conditions. This delay allows the animal to reduce its activity during the unfavorable midday, when temperatures are highest. Most interestingly, this phenotype is easily visible even without temperature changes, underscoring the importance of light as a major entrainment cue (Schlichting, 2019).

A central finding is that flies lacking the compound eyes show an entrainment deficit, i.e., they have an advanced E peak under long-day conditions. Similarly, they show a reduction in M peak amplitude; this is likely due to a failure to properly activate PDF-expressing neurons. Using rhodopsin mutants and by manipulating specific photoreceptors using the GAL4/UAS system, only R8 of the compound eyes appears essential for this summer day response; R8 was previously implicated in the adaptation to nature-like light conditions (Schlichting, 2019).

Notably, even flies lacking all compound eyes significantly delay their E peak timing under long photoperiod conditions but to a much smaller extent. This indicates that other photoreceptors also contribute to the E peak delays under these conditions. One likely candidate is the HB-eyelet. Recent work has shown that this photoreceptor contributes to delaying the E peak under high-light-intensity conditions [48]. Similar mechanisms might apply under long days, which is supported by the strongly advanced E peak when rh5-GAL4 and rh6-GAL4-positive neurons, which includes the HB eyelets, were silenced or ablated (Schlichting, 2019).

lLNv arbors in the optic lobe are in close proximity to R8 termini, where they most likely interact via cholinergic interneurons in addition to the accessory medulla, which was recently shown to be important for light-evoked responses of the lLNvs. This interaction results in a change of neuronal bursting behavior and hence neuropeptide release. Indeed, this study shows that release of PDF from the lLNvs is necessary and sufficient for proper long-day adaptation (Schlichting, 2019).

These results are surprising given a recently published study on how the visual system is connected to the clock neuron network vv. It shows that the visual system can activate a broad spectrum of lateral and dorsal neurons; they include sLNvs, lLNvs, ITP+ LNds, and DN2s, among others. Ablation of PDF neurons left the other neurons responsive to visual input, suggesting a parallel model for clock synchronization, i.e., information from the visual system can be directly transferred to independent classes of clock neurons rather than only via PDF. This new pathway might be involved in the residual delay of E activity in pdf01 flies under long photoperiods, suggesting a potential PDF-independent contribution to long-day adaptation (Schlichting, 2019).

PDF stimulates different adenylate-cyclases and increases cAMP, which leads to the stabilization of PER and TIM and consequently a longer period or phase delay. Therefore, one view is that removing the compound eyes decreases PDF release from the lLNvs and phase advances the molecular clock in downstream target neurons like the LNds. This newly discovered 'visual system to LNd pathway' might also enhance CRY-mediated photoentrainment: CRY was shown to activate neurons upon stimulation, similar to the newly identified light activation of clock neuron pathway. Additional activation of the E cells could therefore contribute to the kinetics of TIM degradation, which was recently shown to be important for the phase advance of E activity under long-day conditions (Schlichting, 2019).

An intriguing inference of this work is that the principal targets of PDF must change with the environmental conditions. Previous work established the sLNvs as essential for DD rhythmicity, and recent work shows that these neurons are tightly coupled to the dorsal clock neurons in DD: speeding up the PDF neurons forced the DN1s to follow the short period of the sLNvs. In LD, however, this connection is much weaker, and the cell-type-specific CRISPR/Cas9 knockout strategy shows that it is the PDF-expressing lLNvs that communicate with the LNd neurons. The data show that the lLNv to LNd connection is important in LD conditions but does not affect DD behavior (Schlichting, 2019).

Importantly, the data not only indicate a qualitative shift of PDF targets between DD and LD but also suggest a quantitative shift of dominance, depending on photoperiod or the time of light exposure. In DD, the sLNvs are necessary for rhythmic behavior and show robust cycling in PER oscillations, whereas the lLNvs lose PER rhythms as early as the second day of DD. In equinox conditions, both groups may be relevant: PDF from either the sLNvs or lLNvs is sufficient for WT behavior, and only knockdown in both sets of neurons is able to reproduce the pdf⁰¹ mutant phenotype. In long photoperiods, however, PDF from the lLNvs is necessary and sufficient for proper entrainment, whereas the sLNvs do not contribute to E peak timing. The data therefore point to a profound circuit switch in response to photoperiod, analogous to the neurotransmitter switching that occurs in the mammalian paraventricular nucleus in response to long photoperiods (Schlichting, 2019).

A similar circuit reorganization might also occur in the principal mammalian brain clock neuron location, the suprachiasmatic nucleus (SCN). Light information from the visual system is transferred to cells in the ventral part of the SCN, which expresses VIP. VIP functions similarly to Drosophila PDF and is not only important for communication between different parts of the SCN but also essential for seasonal encoding. This is because VIP knockout mice show no change in peak width as measured by in vivo electrophysiological recordings in response to entrainment to different photoperiods]. This suggests that VIP is not only involved in relaying light information beyond the ventral SCN but also in the response to light duration as shown in this study for PDF in Drosophila. It will be interesting to see whether different VIP-expressing SCN neurons are involved in this response (Schlichting, 2019).

In Drosophila, the clock that controls rest-activity rhythms synchronizes with light-dark cycles through either the blue-light sensitive Cryptochrome (Cry) located in most clock neurons, or rhodopsin-expressing histaminergic photoreceptors. This study shows that, in the absence of Cry, each of the two histamine receptors Ort and HisCl1 contribute to entrain the clock whereas no entrainment occurs in the absence of the two receptors. In contrast to Ort, HisCl1 does not restore entrainment when expressed in the optic lobe interneurons. Indeed, HisCl1 is expressed in wild-type photoreceptors and entrainment is strongly impaired in flies with photoreceptors mutant for HisCl1. Rescuing HisCl1 expression in the Rh6-expressing photoreceptors restores entrainment but it does not in other photoreceptors, which send histaminergic inputs to Rh6-expressing photoreceptors. The results thus show that Rh6-expressing neurons contribute to circadian entrainment as both photoreceptors and interneurons, recalling the dual function of melanopsin-expressing ganglion cells in the mammalian retina (Schlichting, 2019).

REGULATION

Functional PDF signaling in the Drosophila circadian neural circuit is gated by Ral A-dependent modulation

The neuropeptide PDF promotes the normal sequencing of circadian behavioral rhythms in Drosophila, but its signaling mechanisms are not well understood. This study reports daily rhythmicity in responsiveness to PDF in critical pacemakers called small LNvs. There is a daily change in potency, as great as 10-fold higher, around dawn. The rhythm persists in constant darkness and does not require endogenous ligand (PDF) signaling or rhythmic receptor gene transcription. Furthermore, rhythmic responsiveness reflects the properties of the pacemaker cell type, not the receptor. Dopamine responsiveness also cycles, in phase with that of PDF, in the same pacemakers, but does not cycle in large LNv. The activity of RalA GTPase in s-LNv regulates PDF responsiveness and behavioral locomotor rhythms. Additionally, cell-autonomous PDF signaling reversed the circadian behavioral effects of lowered RalA activity. Thus, RalA activity confers high PDF responsiveness, providing a daily gate around the dawn hours to promote functional PDF signaling (Klose, 2016).

Dual PDF Signaling Pathways Reset Clocks Via TIMELESS and Acutely Excite Target Neurons to Control Circadian Behavior

Molecular circadian clocks are interconnected via neural networks. In Drosophila, Pigment-Dispersing Factor (PDF) acts as a master network regulator with dual functions in synchronizing molecular oscillations between disparate PDF⁺ and PDF^- circadian pacemaker neurons and controlling pacemaker neuron output. Yet the mechanisms by which PDF functions are not clear. This study has demonstrated that genetic inhibition of protein kinase A (PKA) in PDF^- clock neurons can phenocopy PDF mutants, while activated PKA can partially rescue PDF receptor mutants. PKA subunit transcripts are also under clock control in non-PDF DN1p neurons. To address the core clock target of PDF, per was rescued in PDF neurons of arrhythmic per⁰¹ mutants. PDF neuron rescue induced high amplitude rhythms in the clock component Timeless (Tim) in per-less DN1p neurons. Complete loss of PDF or PKA inhibition also results in reduced Tim levels in non-PDF neurons of per⁰¹ flies. To address how PDF impacts pacemaker neuron output, PDF was focally applied to DN1p neurons and was found to acutely depolarize and increase firing rates of DN1p neurons. Surprisingly, these effects are reduced in the presence of an adenylate cyclase inhibitor, yet persist in the presence of PKA inhibition. Evidence is provided for a signaling mechanism (PKA) and a molecular target (Tim) by which PDF resets and synchronizes clocks; PDF exhibits an acute direct excitatory effect on target neurons to control neuronal output. The identification of Tim as a target of PDF signaling suggests it is a multimodal integrator of cell autonomous clock, environmental light, and neural network signaling. Moreover, these data reveal a bifurcation of PKA-dependent clock effects and PKA-independent output effects. Taken together, these results provide a molecular and cellular basis for the dual functions of PDF in clock resetting and pacemaker output (Seluzicki, 2014).

Transcriptional Regulation

One of the most interesting questions in circadian biology is how a molecular cycle is translated into time of day information for the behaving organism. pdf expression is regulated by the Drosophila clock and requires cycling vrille expression. pdf encodes a neuropeptide expressed in the axons of the pacemaker cells, and these projections connect the LNs with target cells in the dorsal brain. PDF protein has been shown to accumulate in the LN axons with a circadian rhythm. The period of this rhythm is shortened by the per^S mutation, and continuous accumulation of PDF in the dorsal brain is associated with arrhythmia and a variety of period changes in adult locomotor activity. PDF mRNA levels do not cycle in wild-type flies. Since continuous expression of vri suppresses PDF protein accumulation without affecting accumulation of pdf mRNA, cycling Vri expression in wild-type Drosophila is likely to contribute to the observed cycling of Pdf protein. Vri may affect Pdf levels by specifying rhythmic expression of a factor involved in translation, maturation, stabilization, transport, or release of the neuropeptide (Blau, 1999 and references therein).

All of these observations point to a likely role for Pdf in coupling a molecular clock to timed behavior; this study has demonstrated that vri conveys essential regulatory signals from the clock to Pdf. There is also evidence that Pdf can in turn influence function of the clock. In the cockroach, microinjection of PDF produces time-dependent shifts in the phase of the locomotor activity rhythm (Petri, 1997). The magnitude of these phase shifts (up to 4 hr) is similar to that produced by light (Petri, 1997). This indicates that a transient change in Pdf level will cause a stable change in molecular components of a clock that regulates behavior in at least some insects. Possibly, the novel pathway of per and tim suppression observed in V2 and V3 Drosophila lines is a direct consequence of eliminating Pdf (Blau, 1999).

Regulation of the Drosophila pigment-dispersing factor (pdf) gene products was analyzed in wild-type and clock mutants. Mutations in the transcription factors Clock and Cycle severely diminish pdf RNA and neuropeptide (PDF) levels in a single cluster of clock-gene-expressing brain cells, called small ventrolateral neurons (s-LN_vs). This clock-gene regulation of specific cells does not operate through an E-box found within pdf regulatory sequences. PDF immunoreactivity exhibits daily cycling, but only within terminals of axons projecting from the s-LN_vs. This posttranslational rhythm is eliminated by period or timeless null mutations, which do not affect PDF staining in cell bodies or pdf mRNA levels. Therefore, within these chronobiologically important neurons, separate elements of the central pacemaking machinery regulate pdf or its product in novel and different ways. Coupled with contemporary results that show a pdf-null mutant to be severely defective in its behavioral rhythmicity, the present results reveal PDF as an important circadian mediator whose expression and function are downstream of the clockworks (Park, 2000).

To assess the effects of clock mutations on pdf expression, the normal cellular distribution of the Drosophila gene's native products were examined. By in situ hybridization, the expression pattern of pdf mRNA has been shown to be similar to that determined with anti-crab-PDH. There are four positive cells in each larval brain hemisphere; these persist into adulthood and become the small ventrolateral neurons (s-LN_vs), whose neurites project into a dorsal region of the adult brain. Four large ventrolateral neurons (l-LN_vs) also express pdf; these emerge during metamorphosis and send projections into the optic lobe and across the brain midline. Larvae and adults also contain pdf mRNA in the posterior extremity of the CNS (Park, 2000).

Northern blots reveal no daily rhythm of pdf mRNA abundance, but they could have failed to detect pdf mRNA cycling in a subset of the cells. Thus temporal in situ hybridizations were performed; neither category of pdf-expressing neurons exhibit systematic fluctuations in signal intensities. Therefore, there is no pdf mRNA rhythm for clock mutations to affect (Park, 2000).

Anti-Drosophila PDF antibodies give cell labeling identical to that obtained by in situ hybridization. Neither method leads to marking of cells in the dorsal brain of adults that are stained by anti-crab-PDH. This indicates that the dorsally located antigen is cross-reacting material and does not have to be considered in terms of effects of clock mutations on pdf expression (Park, 2000).

Expression of pdf in the arrhythmic Clk^Jrk mutant has been found to be strikingly abnormal. In Clk^Jrk brains, neither pdf mRNA nor PDF is detectable in larval LN cells and in the s-LN_vs of adults. The same defects were observed in mutant animals heterozygous for Clk^Jrk and a deletion of the locus. These results suggest that Clk is required for pdf transcription, although only in certain cells: the larval LNs and the s-LN_vs into which they develop. Dorsally projecting axonal processes arising from the s-LN_v cells terminate near the calyx of the dorsal-brain mushroom body. In accord with the absence of perikaryal s-LN_v immunoreactivity, these projections are absent from Clk^Jrk brains. In contrast, expression in the l-LN_vs and abdominal-ganglionic cells of adults is apparently unaffected by Clk^Jrk and D); this includes normal staining of centrifugal and interhemispheric projections within the fly's head. However, certain features of projections from l-LN_v cells are aberrant in Clk^Jrk. Approximately 50% of the mutant brains showed abnormal projections; in others, one or two axons from this region project further and irregularly toward a dorsal or median region of the brain. None of these projections is similar to the more dorsal-reaching projections in the brains of wild-type adults (Park, 2000).

Because the Cyc protein cooperates with Clk in their transcriptional-activation roles, pdf expression was examined in cycle mutants. The effects were similar to but less severe than those caused by Clk^Jrk. Most of the larval LNs homozygous for either of two cyc⁰ mutations show much weaker expression of both mRNA and peptide, as compared with wild type, but the mutant expression levels are variable even within a single brain hemisphere: some cells contained signal, whereas others are extremely difficult to detect. The numbers of antibody-stained s-LN_vs in cyc⁰ adults are well above zero, compared with the elimination of such signals in Clk^Jrk flies. Numbers of l-LN_v cells in the brains of cyc-mutated adults are normal, similar to the results obtained in the Clk^Jrk background. About 25% of the adult cyc⁰ brains exhibit an abnormal dorsal projection. In approximately 30% of these mutant specimens, the projections are asymmetric within a single individual: one hemisphere can contain a bundle of dorsally projected axons; but in the contralateral hemisphere, only one or two axons project into the dorsal brain. In other cyc⁰ adults, axons project irregularly into a median brain region (Park, 2000).

The major conclusions from examining pdf expression in the Clock and cycle mutants are that (1) both genes appear to be positive regulators of pdf RNA levels but only in the s-LN_vs and their larval precursors; (2) the effects of Clk^Jrk are stronger than those of the cyc⁰ mutations; and (3) there are developmental defects, because PDF-containing processes in the adult CNS are aberrant in both types of mutants (Park, 2000).

Do Clk and Cyc activate pdf transcription directly? If that is the case, there could be an E-box in this gene's regulatory region. Indeed, within a 2.4-kb segment 5' to the pdf ORF a CACGTG sequence ~1.4 kb upstream of the transcription-start site has been found. The 2.4-kb DNA fragment was fused to the (yeast) GAL4 gene; transgenic strains were generated and crossed to flies carrying UAS-lacZ. The doubly transgenic progeny show faithful beta-galactosidase-reported expression of pdf. To determine whether the E-box is important for the pdf's transcriptional activation, further transgenics were generated. Deletions missing either half or all of the E-box are sufficient to drive brain expression indistinguishable from that observed in wild type. Interestingly, the smallest 5'-flanking region examined mediates the normal brain pattern but does not lead to abdominal-ganglionic expression in the larval CNS. That the influences on pdf expression of Clock and cycle do not operate through a circadian E-box, and thus seem to be indirect, is consistent with the lack of pdf mRNA cycling and Clock/cycle-independent expression in the l-LN_v cells (Park, 2000).

No effect of a period-null mutation on pdf mRNA levels had been detectable in previous Northern blottings. Neither per⁰¹ nor a timeless-null mutation affects the RNA's abundance, by Northern blottings and by in situ hybridizations. To search further for regulation by per or tim, adult brains were stained with anti-PDH at different times of day and night. Strikingly, nerve terminals in a dorsal region of the central brain exhibit rhythms of anti-PDH-mediated staining. The neurites that terminate in this region project from the s-LN_v cells. In an LD cycle, the peak and trough times for the nerve-terminal cycling are 1 h after lights-on and lights-off, respectively. Staining levels in the perikarya of s-LN_vs exhibit some fluctuations but no regular pattern. The adult-specific, larger PDF neurons also exhibit no appreciable cycling of anti-PDH-mediated staining, either in l-LN_v cell bodies or in the termini of their neurites that ramify over the surface of the medulla optic lobe (Park, 2000).

The dorsal-brain, nerve-terminal cycling persists in constant darkness with an ~24-h period in wild type. In that condition the cycle duration is shortened to ~20 h by the per^S mutation, which causes behavioral periodicities to be about 5 h shorter than normal. In the dorsal brains of the per⁰¹ null mutant, nerve-terminal cycling is abolished, and the signal strengths are very low. However, the immunohistochemical procedure performed on these brain sections is not very sensitive. Therefore, a quantitative fluorescence method was used, the better to judge PDF staining intensities in whole-mounted brains. At the peak and trough time-points, nerve-terminal signals in wild type are again higher in the early morning compared with the early night. This temporal difference is not observed in the dorsal brains of the arrhythmic per⁰¹ and tim⁰¹ mutants. In per⁰¹, the staining intensities at both times are nearly identical and at levels intermediate between the per⁺ peaks and troughs. In tim⁰¹, the PDF terminal signals are also the same at the two time-points but significantly higher than in tim⁺ and. The mutational effects of these clock genes on daily fluctuations of PDF abundance at certain nerve terminals indicate that an aspect of this peptide's regulation is, in one way clock controlled, and in another was posttranslationally regulated (Park, 2000).

Morning and evening peaks of activity rely on different clock neurons of the Drosophila brain

In Drosophila, a 'clock' situated in the brain controls circadian rhythms of locomotor activity. This clock relies on several groups of neurons that express the Period (Per) protein, including the ventral lateral neurons (LN_vs), which express the Pigment-dispersing factor (PDF) neuropeptide, and the PDF-negative dorsal lateral neurons (LN_ds). In normal cycles of day and night, adult flies exhibit morning and evening peaks of activity; however, the contribution of the different clock neurons to the rest-activity pattern remains unknown. Targeted expression of Per was used to restore the clock function of specific subsets of lateral neurons in arrhythmic per⁰ mutant flies. Per expression restricted to the LN_vs only restores the morning activity, whereas expression of PER in both the LN_vs and LN_ds also restores the evening activity. This provides the first neuronal bases for 'morning' and 'evening' oscillators in the Drosophila brain. Furthermore, the LN_vs alone can generate 24 h activity rhythms in constant darkness, indicating that the morning oscillator is sufficient to drive the circadian system (Grima, 2004).

PDF function in resetting signal between Drosophila pacemakers synchronizes morning and evening activity

Daily rhythms of physiology and behaviour are precisely timed by an endogenous circadian clock. These include separate bouts of morning and evening activity, characteristic of Drosophila melanogaster and many other taxa, including mammals. Whereas multiple oscillators have long been proposed to orchestrate such complex behavioural programs, their nature and interplay have remained elusive. By using cell-specific ablation, it has been shown that the timing of morning and evening activity in Drosophila derives from two distinct groups of circadian neurons: morning activity from the ventral lateral neurons that express the neuropeptide PDF, and evening activity from another group of cells, including the dorsal lateral neurons. Although the two oscillators can function autonomously, cell-specific rescue experiments with circadian clock mutants indicate that they are functionally coupled (Stoleru, 2004).

The biochemical machinery that underlies circadian rhythms is conserved among animal species and drives self-sustained molecular oscillations and functions, even within individual asynchronous tissue-culture cells. Yet the rhythm-generating neural centres of higher eukaryotes are usually composed of interconnected cellular networks, which contribute to robustness and synchrony as well as other complex features of rhythmic behaviour. In mammals, little is known about how individual brain oscillators are organized to orchestrate a complex behavioural pattern. Drosophila is arguably more advanced from this point of view: a group of adult brain clock neurons expresses the neuropeptide PDF and controls morning activity (small LN_v cells; M-cells), whereas another group of clock neurons controls evening activity (CRY⁺, PDF^- cells; E-cells). Transgenic mosaic animals were generated with different circadian periods in morning and evening cells. This study shows by behavioural and molecular assays, that the six canonical groups of clock neurons are organized into two separate neuronal circuits. One has no apparent effect on locomotor rhythmicity in darkness, but within the second circuit the molecular and behavioural timing of the evening cells is determined by morning-cell properties. This is due to a daily resetting signal from the morning to the evening cells, which run at their genetically programmed pace between consecutive signals. This neural circuit and oscillator-coupling mechanism ensures a proper relationship between the timing of morning and evening locomotor activity (Stoleru, 2005).

Overexpression of the Tim kinase Shaggy (Sgg; Drosophila GSK3) shortens the period by 3-4 h. Sgg expression was driven in all clock cells by crossing tim-GAL4 with flies carrying an EP element inserted at the Sgg locus (EP1576, referred to as UAS-Sgg). The locomotor activity rhythm of tim-GAL4/UAS-Sgg (timSgg) flies in constant darkness (DD) confirmed previous results, in that the period was about 3 h shorter than that of control flies (Stoleru, 2005).

Sgg was expressed exclusively in LN_v cells by constructing a Pdf-GAL4/UAS-Sgg genotype. The Pdf-GAL4 driver is well characterized and drives gene expression only in two clock-cell groups: the PDF⁺ small LN_v (s-LN_v) cells (that is, M-cells) and the PDF⁺ large LN_v (l-LN_v) cells. The driver is inactive in the CRY⁺PDF^- evening cells. Pdf-GAL4/UAS-Sgg (PdfSgg) flies also manifested a short period. The period shortening was less than that of timSgg flies, probably because of weaker expression from Pdf-GAL4 driver in LN_v cells. Sgg expression from an even weaker driver, cry₁₃-GAL4, did not affect behavioural period (Stoleru, 2005).

A close inspection of the behavioural actograms revealed that the period of evening activity is significantly shorter in PdfSgg flies (with a daily advance of about 2 h). This indicates that the pace of E-cells was accelerated, although the period manipulation was restricted to M-cells. An advanced evening peak, without an increase in E-cell Sgg expression, indicates that the faster M oscillator might be setting the E-cell pace. It is therefore proposed that the PDF⁺ cells influence molecular circadian events within E-cells (Stoleru, 2005).

To investigate this possibility, the molecular period (cycle duration) of each clock-cell group was estimated in these different genotypes: UAS-Sgg (control), timSgg and PdfSgg. Fly brains were analysed by in situ hybridization for tim RNA expression pattern after 4 days in DD, so that a barely detectable daily advance by 2-3 h would result in an aggregate advance of 8-12 h on DD4 (fourth day of DD). Indeed, Sgg overexpression in all clock neurons (timSgg) markedly shifted the interval of high tim mRNA expression on DD4 by about 12 h, from between CT10 and CT18 to before CT6. (CT is the circadian time within a constant-darkness experiment; CT0 is the hour of the last lights-on event.) All neurons expressing clock genes showed a similar temporal pattern, consistent with the expected Sgg-induced period shortening in all clock cells, and with a deterministic relationship between the molecular period and the locomotor activity period (Stoleru, 2005).

However, the PdfSgg tim RNA profiles were strikingly different and unexpected. Whereas the s-LN_v cells showed a roughly 8 h advance in DD4, expected from a period shortening of 2 h per day, the l-LN_v cells showed no appreciable change from those in control flies; that is, their molecular program is apparently unaffected by Sgg overexpression within these cells. Also surprising were the DN1 and DN3 profiles, which showed a roughly 8 h advance, as were the LN_d cells, which were advanced by about 6 h relative to those in control flies. Since PdfSgg flies do not overexpress Sgg in these three cell groups, their molecular programs behave in a non-cell-autonomous manner. Because the E-cells are included within these groups and because the s-LN_v cells (the M-cells) are the only cells with a cell-autonomous program that match the behavioural period of the flies, the M-cells apparently determine the clock pace of these other neuronal groups, including the E-cells (Stoleru, 2005).

The l-LN_v cells and DN2 cells emerged as the only clock-gene-expressing neurons that evaded control of the M-cells and maintained a wild-type-like phase of tim RNA cycling in PdfSgg flies. Because DN2 cells are genotypically wild type in these flies, it is inferred that they oscillate with cell-autonomous properties and are the best candidates for determining the non-cell-autonomous wild-type-like characteristics of the l-LN_v cells. As a consequence there are at least two parallel clock-cell circuits in the Drosophila brain in constant darkness: the M-E circuit controls locomotor activity rhythms and is driven by the M-cells (s-LN_v cells), whereas the DN2-l-LN_v circuit has as yet unknown functions and is driven by the DN2 cells (Stoleru, 2005).

To verify and extend these concepts, a genotype was generated in which the E-cells should run faster than M-cells. By adding the previously described Pdf-GAL80 repressor construct to the tim-GAL4;UAS-Sgg background, Sgg was expected to be overexpressed in all clock neurons with the exception of PDF-expressing cells. As these include the M-cells (s-LN_v cells), they should run more slowly (24 h) than the E-cells (about 21 h). A 'faster takes all' rule predicts that the short-period E-cells will dominate over the normal 24 h M-cells in this genotype and generate a behavioural rhythm of about 21 h. Alternatively, dominant M-cells will give rise to a behavioural period of 24 h despite the faster endogenous oscillator in the E-cells (Stoleru, 2005).

Consistent with a dominant M-cell model was the observation that timSgg/PdfGAL80 flies had an almost wild-type period in DD. The molecular analysis is also consistent, since the s-LN_v cells manifested a wild-type-like program: tim mRNA peaked between CT12 and CT20 on DD4. Despite Sgg overexpression, the LN_d cells, DN1 cells and DN3 cells had a similar and wild-type-like pattern of tim expression. As described above, this indicates that all three cell groups behave non-autonomously and are probably driven by the s-LN_v cells. This result is supported by the anatomical pattern of s-LN_v neuronal processes, which project towards the brain regions populated by LN_d, DN1 and DN3 cells. DN2 cells were again the only Sgg-overexpressing cells in which the phase of tim RNA oscillation corresponded to the predicted accelerated pace. The l-LN_v cells, despite lacking Sgg overexpression (because of the PdfGAL80 transgene), also showed a comparable advance of tim expression. These timSgg/PdfGAL80 results confirm that the s-LN_v cells determine the phase of LN_d, DN1 and DN3 cells and that an independent cellular network includes the DN2 and l-LN_v cells. Because the behavioural period was wild-type-like and paralleled the molecular clock within the s-LN_v cells, the results confirm that these M-cells assign the circadian period in the absence of light cues (Stoleru, 2005).

To confirm the lack of a contribution of DN2/l-LN_v to the E-M network function and to locomotor rhythms, the timSgg/cryGAL80 genotype was also examined. It is similar to the timSgg/PdfGAL80 genotype described above, except that Sgg overexpression is repressed in a wider group of cells. These include most if not all of the E-cells and l-LN_v cells as well as the M-cells. Since DN2 cells are the only clock cells in which cry promoter-driven expression was not detected, it is expected that the faster clock in timSgg/cryGAL80 would be limited to CRY^- cells, including the apparently cell-autonomous DN2 cells (Stoleru, 2005).

Indeed, tim hybridization in situ confirmed that the period of DN2 rhythm was shortened by about 2-3 h per day. The l-LN_v neurons were shifted to about the same extent, which is consistent with the notion that they behave non-cell-autonomously and follow the pace of the DN2 clock program. All other clock cells maintained a pattern similar to that of control flies. Because timSgg/cryGAL80 flies had a normal behavioural period, these results confirm that l-LN_v and DN2 cells do not contribute detectably to locomotor activity rhythms. This conclusion is in agreement with previous results showing that wild-type flies have persistent DD behavioural rhythms, despite protein oscillation idiosyncrasies of the l-LN_v and DN2 cells (Stoleru, 2005).

How does the M-cell (s-LN_v) clock determine the period of E-cells (LN_d cells/DN cells)? Although previous work indicated possible oscillator coupling and a direct effect of LN_v on the transcriptional oscillations of other clock cells, it was difficult to envision how the M-cells could override the intrinsic molecular timing of the E-cells. A second possibility is therefore considered, namely that the E-cells maintain an unaltered intrinsic clock program but receive a daily resetting signal from the M-cells. This model predicts that the timing of the evening activity within every cycle (between two consecutive mornings) reflects the status of the endogenous clock of E-cells, whereas the overall period exhibited by the evening peaks reflects the pace of the M-cell resetting signal (Stoleru, 2005).

To examine this possibility, the different transgenic strains were assayed for their average evening activity phase within a cycle, by using the leading morning peak as a reference and then measuring the average time until the subsequent evening peak. The overall DD period correlated with the genotype of M-cells as expected, but the length of the subjective day (M-E interval) correlated only with the genotype of the E-cells. In control flies with a period of about 24 h, the subjective day was roughly 12 h, similar to the duration of subjective day of PdfSgg. The latter strain features a wild-type-like E-oscillator but a fast, Sgg-expressing M-oscillator and a period of about 22 h. In contrast, timSgg flies express Sgg in both E-cells and M-cells, and both the average length of subjective day and the period (M-M) are reduced. The results indicate that the E-cells run an autonomous clock program whose starting (or ending) points are determined by daily resetting signals from the M-cells (Stoleru, 2005).

A DD unidirectional M ---> E resetting mechanism also predicts that a slower (24 h) M-cell clock and a faster E-cell clock will have a normal morning peak phase but an advanced evening peak phase. To test this prediction, the behavioural outputs of timSgg/PdfGAL80 and timSgg/cryGAL80 flies, which differ only in the genotypes of their E-cells, were compared. Both strains have periods of about 24 h, but the former should give rise to a fast E-cell molecular program, whereas the latter should have an E-clock of 24 h as a result of suppression of Sgg expression (Stoleru, 2005).

Indeed, the evening phase of timSgg/cryGAL80 is similar to that of control flies, and it always occurs about 2.5 h later than that of timSgg/PdfGAL80. The evening phase of timSgg/PdfGAL80 is more similar to that of timSgg, although the latter genotype has a much shorter period than the former. The length of subjective day of timSgg/PdfGAL80 flies further confirms that the evening phase within each cycle is a reflection of the endogenous E-cell rhythm, whereas the period of the cycle (M-M) correlates with the intrinsic M-cell clock (Stoleru, 2005).

These comparisons indicate that the circadian network is modulated by intercellular communication signals, which achieve and maintain circadian coherence -- the proper relationship between morning and evening activity. The dominant M-clock determines the period of the entire system by providing a daily reset signal to the E-clock in darkness and is therefore a true cellular Zeitgeber. Because the M-cells can delay as well as advance E-cells, the resetting signal may be required for E-cell oscillations. The usual candidate for this signal is the M-cell-specific neuropeptide PDF. It contributes to the normal synchrony and/or rhythmicity in constant darkness, with a striking similarity to the mammalian neuropeptide VIP. Moreover, injecting PDF into the cockroach brain causes circadian phase delays. Other principles and/or molecules may also be relevant to the M-E subnetwork, because E-cells can drive clockless M-cells to manifest cyclical behavioural outputs under 12 h light/12 h dark (LD) conditions (Stoleru, 2005).

The l-LN_v and DN2 cells are the two neuronal groups that escape the M-cell reset signal in DD. They constitute a second circadian subnetwork with no apparent effect on locomotor activity rhythms and no known function. The DN2 cells are among the few clock-gene-expressing brain cells in larvae and are also the only clock cells that do not change their morphology after eclosion. Larval DN2 cells are apparently devoid of CRY and manifest anti-phase oscillations of Tim and PER. It is therefore likely that both the DN2 cells and the l-LN_v cells impart circadian regulation to unknown physiological functions relevant to both larvae and adults. More generally, it is expected that the organizational principles of the two subnetworks described in this study will also be relevant to mammalian neuronal networks with important behavioural functions, for example the relationship between different oscillators in the SCN (Stoleru, 2005).

Electrical hyperexcitation of lateral ventral pacemaker neurons desynchronizes downstream circadian oscillators in the fly circadian circuit and induces multiple behavioral periods

Coupling of autonomous cellular oscillators is an essential aspect of circadian clock function but little is known about its circuit requirements. Functional ablation of the pigment-dispersing factor-expressing lateral ventral subset (LN_V) of Drosophila clock neurons abolishes circadian rhythms of locomotor activity. The hypothesis that LN_Vs synchronize oscillations in downstream clock neurons was tested by rendering the LN_Vs hyperexcitable via transgenic expression of a low activation threshold voltage-gated sodium channel. When the LN_Vs are made hyperexcitable, free-running behavioral rhythms decompose into multiple independent superimposed oscillations and the clock protein oscillations in the dorsal neuron 1 and 2 subgroups of clock neurons are phase-shifted. Thus, regulated electrical activity of the LN_Vs synchronize multiple oscillators in the fly circadian pacemaker circuit (Nitabach, 2006).

Understanding the mechanisms for synchronizing multiple independent neural oscillators in circadian circuits is a key issue in circadian biology. This study provides evidence that the excitability state of the LN_V subset of clock neurons plays a critical role in coordinating multiple oscillators in the fly brain. When the LN_Vs are made electrically hyperexcitable by genetically targeted expression of a voltage-gated sodium channel cloned from a halophilic bacterium, NaChBac, transgenic flies exhibit complex free-running behavioral rhythms with multiple periods along with desynchronization of clock protein cycling throughout the pacemaker circuit and disrupted cycling of PDF levels in the dorsomedial terminal projections of the small LN_Vs (sLN_Vs) (Nitabach, 2006).

Anti-PDF immunofluorescence was observed in the dorsomedial terminals of the sLN_Vs in control flies. However, anti-PDF immunofluorescence in the dorsomedial terminals of the sLN_Vs of experimental flies expressing NaChBac in the LN_Vs is maintained at constitutively higher levels. This result is unexpected if PDF release at nerve terminals is the only cellular function influenced by alterations in cellular electrical excitability. Although there remains a formal possibility that NaChBac expression does not cause increased electrical excitability in pacemaker neurons, this is considered highly unlikely because of the robust and opposite effects of NaChBac expression compared with open-rectifier potassium-channel expression on behavior, reciprocal rescue of behavior by coexpression, clock oscillation, and direct electrophysiological recordings of muscle and photoreceptor neurons expressing NaChBac. Furthermore, hyperpolarization of LN_v membrane potential after the targeted expression of open-rectifier potassium channels to these cells causes accumulations of PDF in the cell bodies of the LN_Vs, providing further evidence that membrane potential regulates the rates of synthesis and/or trafficking of PDF as well as release. These results together suggest that regulated electrical excitability of the sLN_V plasma membrane underlies cycling PDF levels in the dorsomedial terminals, and that rendering the sLN_Vs hyperexcitable through NaChBac expression disrupts one or more of the cellular processes (synthesis, trafficking, or release) that determine PDF accumulation in the dorsomedial terminals. It remains unclear whether changes in neuronal membrane excitability directly influences PDF accumulation or whether this is caused by indirect effects via the molecular clock, because PDF accumulation appears to be restricted to pacemaker neurons (Nitabach, 2006).

The behavioral and circuit alterations caused by NaChBac expression in the LN_Vs may be attributable in part to an altered pattern of PDF release or a yet-unidentified neurotransmitter released by the LN_Vs, or to complex circuit properties of the pacemaker circuit. Regulated membrane electrical excitability of other neuropeptide-secreting neurons of the insect nervous system is known to be essential for appropriate control of the temporal patterns of peptide release. PDF may act as an intrinsic coupling signal within the circadian clock circuit that synchronizes multiple oscillators that otherwise free-run independently. This interpretation is consistent with a synchronizing role for PDF proposed on the basis of gradual phase dispersal within the sLN_V subgroup of Pdf⁰¹-null mutant flies in constant darkness. In addition, the results are consistent with the idea that temporally regulated PDF release by the LN_Vs synchronizes the circuit, and are inconsistent with the hypothesis that PDF plays a purely permissive role (Nitabach, 2006).

Recent electrophysiological evidence in another insect suggests a mechanism for PDF- and GABA-mediated synchronization of multiple oscillators of pacemaker circuits (Schneider, 2005). Extracellular multiunit recordings of the candidate circadian neurons in excised preparations of the cockroach accessory medulla exhibit ultradian oscillatory action potential firing that is synchronized by local application of pressure ejected PDF and GABA through glass micropipettes or bath applied GABA (Schneider, 2005). Similarly, circadian neurons in the fly may fire in PDF-regulated assemblies. Although there is as yet insufficient electrophysiological evidence to allow direct comparison of the results in Drosophila with this recent finding in the cockroach, this raises the interesting possibility that NaChBac expression in the Drosophila LN_Vs may result in desynchronized firing of pacemaker neurons throughout the circuit, starting with the LN_Vs themselves. This would be consistent with the biophysical property of NaChBac of low-threshold voltage activation. Interestingly, similar mechanisms for oscillator coupling at the circuit level may also be important in mammals. GABA also modulates phase coupling between the ventral and dorsal oscillators in brain slices prepared from the rat SCN (Nitabach, 2006).

The behavioral results confirm that the Drosophila circadian control circuit contains multiple clocks capable of oscillating independently and capable of independently controlling the pattern, but not the amount, of locomotor activity. They further indicate that properly regulated electrical excitability of the LN_Vs (and perhaps of particular importance, the LN_Vs) is required to synchronize these multiple clocks throughout the pacemaker neuronal circuit. The synchronization of multiple oscillators appears to be necessary to generate coherent single-period behavioral rhythms (Nitabach, 2006).

The reciprocal suppression by NaChBac of the arrhythmicity induced by Kir2.1, and by Kir2.1 of the complex rhythmicity induced by NaChBac, strongly supports the interpretation that NaChBac and Kir2.1 have opposite effects on the electrical excitability of the LN_Vs, with Kir2.1 decreasing excitability and NaChBac increasing excitability. When expressed individually in the LN_Vs, K⁺ channels and Na⁺ channels have opposite behavioral effects: hyperpolarizing K⁺-channel expression results in arrhythmic behavior, whereas depolarizing Na⁺-channel expression results in hyper-rhythmic behavior. The coexpression of these two channel types together results in functional reciprocal compensation, yielding nearly normal behavior (Nitabach, 2006).

In a previous studies, LN_V membrane potential was manipulated to be hypoexcitable through the targeted expression of modified open-rectifier or inward-rectifier potassium channels (Nitabach, 2002). This caused behavioral arrhythmicity and cell autonomous dampening of the free-running molecular clock in LN_V neurons in constant darkness, along with no discernable changes in the cycling of the molecular clock in downstream pacemaker neuronal subgroups at circadian day 2. Those results are consistent with the findings that desynchrony of downstream cell groups does not become apparent in pdf⁰¹-null mutant flies until 2 d in constant darkness. In the present study, LN_V hyperexcitability induces trans-synaptic changes in the free-running temporal pattern of clock protein accumulation in the dorsal neuron subgroups DN1 and DN2. Thus, the DN neuronal groups appear to be functionally downstream of the LN_V neurons in the pacemaker circuit. In negative control flies, the DN1s oscillate in phase with the sLN_Vs and LN_Ds, maintaining synchrony on both days 2 and 5 after release into constant darkness from a diurnal 12 h light/dark entraining regime, whereas the DN2s gradually advance from synchrony in 12 h light/dark to a 12 h phase difference by circadian day 5. The DN2s of control flies exhibit peak PDP1 accumulation at CT14 on day 2 in constant darkness and at CT10-CT14 on day 5 in constant darkness. This gradual shift of DN2 PDP1 oscillation from synchrony with the other cell groups in LD to a 12 h phase advance after 5 d in constant darkness is consistent with observations of DN2 PER cycling. In pdf>NaChBac1 flies expressing NaChBac in the LN_Vs, the DN1s exhibit a PDP1 molecular peak 8 h earlier than control flies on day 2 in constant darkness, and by circadian day 5 this peak has significantly damped and an additional significant peak has appeared at CT22. The DN2s of pdf>NaChBac1 flies exhibit a peak of PDP1 accumulation at CT14 on day 2 in constant darkness, in phase with control flies; by day 5 in constant darkness they peak at CT6, 4–8 h earlier than in controls. This phase shift suggests that the DN2 molecular oscillator of pdf>NaChBac1 flies is running faster than that of control flies. These differences in the temporal pattern of PDP1 accumulation in the DN1s and DN2s induced by NaChBac expression in the LN_Vs indicate that properly regulated electrical activity is required for normal patterns of molecular oscillation in these dorsal cell groups (Nitabach, 2006).

The DN2s may be capable of independently driving behavioral outputs, and are possibly the cellular substrate for the ~22 h short-period component of the complex behavioral rhythmicity exhibited by flies expressing NaChBac in the LN_Vs. The cellular substrates for the ~25.5 h long-period component are likely to reside in other cells within the circuit. In control pdf>TM3 flies, robust free-running PER oscillation is observed in the sLN_V,LN_D, and DN1 neurons after 5 d in constant darkness, with trough levels of PER in the second half of subjective day. The differences in the spatiotemporal pattern of PER accumulation induced by NaCh-Bac expression in the LN_Vs confirm, as indicated by the effects on PDP1 accumulation, that hyperexcitation of electrical activity in the LN_Vs causes desynchronization of the coupling and phase of molecular oscillation in dorsal clock neurons (Nitabach, 2006).

Multiple oscillators are distributed throughout the pacemaker circuit in Drosophila. The present study confirms and extends evidence for multiple oscillators in the pacemaker circuit in Drosophila. The independent oscillators driving the multiple period components of the behavioral rhythms that were observed do not appear to correspond directly to the 'morning' and 'evening' oscillators, which have been localized to the LN_Vs and LN_Ds, respectively. The current results emphasize that the activity of the LN_Vs controls the synchronization of independent oscillators throughout the pacemaker circuit. The normal pattern of DN1 and DN2 clock oscillation requires properly regulated electrical excitability of the LN_Vs. Further, the results suggest that the DN2s, and at least some other cell groups, possess independent output pathways to the downstream locomotor circuitry (Nitabach, 2006).

This study introduces a novel method for inducing electrical hyperexcitability in neurons of interest by the expression of the low-threshold voltage-gated sodium channel NaChBac. This method is likely to be useful for the analysis of other neural circuits. In another study (Luan, 2006), the utility of the NaChBac channel for enhancing excitability in other neurons has also been demonstrated. Targeted expression of ion channel subunits in vivo provides a powerful means for precisely perturbing neuronal membrane excitability to probe the role of activity on neuronal development and function. Initial methods to exogenously regulate electrical excitability in neurons in vivo have used potassium channel expression to electrically silence neurons. Exogenous manipulation of electrical excitability within specific Drosophila neurons can be combined with finer parsing of neural circuits using GAL80 and other genetic approaches (Nitabach, 2006).

This study has shown that aberrations of electrical excitability in Drosophila neurons, either hyperexcitability induced by NaChBac or hypoexcitability induced by Kir2.1, can be rescued by coexpression of an ion channel with an opposite effect on excitability. This provides reason to believe that such an approach to neurological disorders of aberrant electrical activity such as epilepsy might indeed be feasible (Nitabach, 2006).

PDF cells are a GABA-responsive wake-promoting component of the Drosophila sleep circuit

Daily sleep cycles in humans are driven by a complex circuit within which GABAergic sleep-promoting neurons oppose arousal. Drosophila sleep has recently been shown to be controlled by GABA, which acts on unknown cells expressing the Rdl GABAA receptor. This study has identified the relevant Rdl-containing cells as PDF-expressing small and large ventral lateral neurons (LNvs) of the circadian clock. LNv activity regulates total sleep as well as the rate of sleep onset; both large and small LNvs are part of the sleep circuit. Flies mutant for pdf or its receptor are hypersomnolent, and PDF acts on the LNvs themselves to control sleep. These features of the Drosophila sleep circuit, GABAergic control of onset and maintenance as well as peptidergic control of arousal, support the idea that features of sleep-circuit architecture as well as the mechanisms governing the behavioral transitions between sleep and wake are conserved between mammals and insects (Parisky, 2008).

Using a variety of mutants and novel genetic strategies to manipulate chronic and acute circuit activity, this study has shown that a small set of circadian clock cells in Drosophila has a critical role in the GABAergic initiation and maintenance of sleep. New genetic tools (dnATPase, ShawRNAi), were developed that allow an increase in the chronic response of neurons to their endogenous inputs. This adds greatly to the arsenal of activity-manipulating tools, most of which suppress firing or neurotransmitter release. Bidirectional manipulation of activity provides much more information about circuit function and dynamics. The utility was demonstrated of a new tool for acute activity manipulation (dTrpA1), which can be used on small numbers of neurons deep within the fly brain. The data suggest a model in which the pdf-GAL4-positive large LNvs (l-LNvs) translate light inputs (and perhaps other arousal signals) into wakefulness. The release of PDF from these cells is required, and l-LNv PDF signals to the smaller s-LNvs. The data demonstrating somnolence after downregulation of PDFR in LNvs indicates that s-LNvs participate in sleep control, although experiments in which they have been ablated suggest that they are not be the only sleep-relevant l-LNv targets. PDF signaling to PDFR-expressing neurons outside the clock that directly control activity is likely to be important. GABA may modulate the ability of LNvs to suppress sleep by acting on either or both s- and l-LNvs (Parisky, 2008).

In mammals, the role of the circadian clock in sleep is not completely understood. It is nonetheless clear that there are genetic (e.g., familial advance sleep phase syndrome) and environmental (e.g., jet-lag, shift work) conditions that disrupt sleep despite primarily affecting the circadian rhythms. The clock has been shown to regulate both when an animal sleeps and how much sleep occurs. The current consensus view is that the mammalian clock is primarily wake-promoting, acting along with the homeostatic sleep drive to shape sleep over the day and night (Parisky, 2008).

The data indicate that in flies PDF and the circadian LNvs more generally regulate both the maintenance of sleep as well as the ability of flies to respond to the wake-promoting effects of light. Although these effects recall the role of the mammalian SCN in sleep regulation, there are few prior links between the Drosophila circadian clock and the regulation of fly sleep. The almost complete elimination of the difference in total sleep between subjective day and subjective night in the pdf⁰¹ background adds substantially to this connection, i.e., light regulation of sleep appears to be substantially circadian clock-mediated Therefore, the contribution of the circadian machinery and fly brain clock circuitry to the control of sleep will probably parallel the important role of the mammalian circadian clock and the SCN in sleep regulation (Parisky, 2008).

PDF neurons have been recently shown to be light-responsive, like some neurons of the mammalian SCN. The l-LNvs also act as the dawn photoreceptor for the clock, sending a reset signal each morning to the rest of the clock. There is also good evidence that fly cryptochrome responds directly to light in addition to influencing circadian timekeeping, and a cry mutant substantially decreases the PDF neuron acute light response. Therefore, some of the waking effects described in this study probably reflect a role of PDF cells on acute processes involving light stimulation. Indeed, the phenotypes of flies without PDF or with decreased LNv neuronal excitability resemble some of the acute effects of the loss of orexin/hypocretin in narcoleptic mice. PDF neurons are also regulated by GABAergic inputs, analogous to those from the basal forebrain that regulate orexin/hypocretin neurons (Parisky, 2008).

Despite these similarities, there are also important organizational differences between systems. Most notable is the wide distribution of sleep circuitry in mammals. There are for example many targets of sleep-promoting GABAergic neurons, and the role of the circadian clock may be largely modulatory. The sleep circuitry of flies is almost certainly more circumscribed and simpler. Indeed, the surprisingly large effects of manipulating Rdl in the 16 LNvs argue that they are a principal target of sleep promoting GABAergic neurons and constitute part of the 'core' sleep circuitry. The fact that activation of a subset of these cells, the l-LNvs, has an effect on sleep homeostasis, further suggests that these cells sit at the heart of the sleep circuit. The fly sleep circuitry may therefore have condensed mammalian stimulatory systems (e.g., histaminergic, cholinergic and adrenergic, as well as orexin) into a simpler and more compact region, which may even largely coincide with the sixteen PDF cells of the circadian circuit (Parisky, 2008).

A limited number of other fly brain regions have been proposed to contribute to fly sleep. Manipulations of a broad set of peptidergic (PHM⁺) cells indicate that peptidergic neurons other than PDF neurons are wake promoting. An attractive hypothesis is that some these other peptidergic cells reside in the pars intercerebralis, a group of neurohumoral cells shown to an important sleep output center. The targets of these cells may even overlap with the targets of LNvs, e.g. the ellipsoid bodies. The PDFR is a class II G-protein coupled receptor and is fairly promiscuous: PDF is the highest affinity ligand, but this receptor is also activated by DH₃₁ and PACAP-38. Since peptidergic modulation may occur by 'volume' transmission instead of by direct synaptic contact, both LNv peptides and peptides from the pars could together affect this motor center to regulate sleep and activity. The role of the pars may be to inform the sleep generation machinery about nutritional and metabolic state, i.e., animals undergoing starvation exhibit hyperlocomotor activity that is believed to be evolutionarily useful as a method for finding food, and alteration of this pars-generated locomotor program affects sleep. The role of l-LNvs is clearly different from that of other PHM+ neurons, and their unique involvement in homeostatic sleep suggests they are central to sleep control (Parisky, 2008).

The only other brain region that has been implicated in Drosophila sleep regulation is the paired structure known as the mushroom bodies. These studies showed that GAL4-driven manipulation of signaling or of neurotransmitter release in this neuropil had complex effects on sleep, not inconsistent with a modulatory role for this sensory integration center. The exact mechanism of these effects is not clear, however, especially since all of the mushroom body GAL4 lines that were examined in this study also express in multiple subsets of clock cells (Parisky, 2008).

The small circuit this study describes presents a tractable model system for understanding the circuit-level control of sleep, the relationship between homeostatic and circadian control as well as the dynamics of sleep-wake transitions; the latter are critical to an understanding of episodic and age-related insomnia (Parisky, 2008).

Tip60 HAT activity mediates APP induced lethality and apoptotic cell death in the CNS of a Drosophila Alzheimer's disease model

Tip60 is a histone acetyltransferase (HAT) enzyme that epigenetically regulates genes enriched for neuronal functions through interaction with the amyloid precursor protein (APP) intracellular domain. However, whether Tip60 mediated epigenetic dysregulation affects specific neuronal processes in vivo and contributes to neurodegeneration remains unclear. This study shows that Tip60 HAT activity mediates axonal growth of the Drosophila pacemaker cells, termed small ventrolateral neurons (sLNvs), and their production of the neuropeptide pigment dispersing factor (PDF) that functions to stabilize Drosophila sleep-wake cycles. Using genetic approaches, loss of Tip60 HAT activity in the presence of the Alzheimer's disease (AD) associated amyloid precursor protein (APP) was shown to affect PDF expression and causes retraction of the sLNv synaptic arbor required for presynaptic release of PDF. Functional consequence of these effects is evidenced by disruption of sleep-wake cycle in these flies. Notably, overexpression of Tip60 in conjunction with APP rescues these sleep-wake disturbances by inducing overelaboration of the sLNv synaptic terminals and increasing PDF levels, supporting a neuroprotective role for dTip60 on sLNv growth and function under APP induced neurodegenerative conditions. These findings reveal a novel mechanism for Tip60 mediated sleep-wake regulation via control of axonal growth and PDF levels within the sLNv encompassing neural network and provide insight into epigenetic based regulation of sleep disturbances observed in neurodegenerative diseases like Alzheimer's disease (Pirooznia, 2012).

Chromatin remodeling through histone-tail acetylation is critical for epigenetic regulation of transcription and has been recently identified as an essential mechanism for normal cognitive function. Altered levels of global histone acetylation have been observed in several in vivo models of neurodegenerative diseases and are thought to be involved in the pathogenesis of various memory related disorders. Chromatin acetylation status can become impaired during the lifetime of neurons through loss of function of specific histone acetyltransferases (HATs) with negative consequences on neuronal function. In this regard, the HAT Tip60 is a multifunctional enzyme involved in a variety of chromatin-mediated processes that include transcriptional regulation, apoptosis and cell-cycle control, with recently reported roles in nervous system function. Previous work has demonstrated that Tip60 HAT activity is required for nervous system development via the transcriptional control of genes enriched for neuronal function. Tip60 HAT activity controls synaptic plasticity and growth as well as apoptosis in the developing Drosophila central nervous system (CNS). Consistent with these findings, studies have implicated Tip60 in pathogenesis associated with different neurodegenerative diseases. The interaction of Tip60 with ataxin 1 protein has been reported to contribute to cerebellar degeneration associated with Spinocerebellar ataxia (SCA1), a neurodegenerative disease caused by polyglutamine tract expansion. Tip60 is also implicated in Alzheimer's disease (AD) via its formation of a transcriptionally active complex with the AD associated amyloid precursor protein (APP) intracellular domain (AICD). This complex increases histone acetylation and co-activates gene promoters linked to apoptosis and neurotoxicity associated with AD. Additionally, misregulation of certain putative target genes of the Tip60/AICD complex has been linked to AD related pathology. These findings support the concept that inappropriate Tip60/AICD complex formation and/or recruitment early in development may contribute or lead to AD pathology via epigenetic misregulation of target genes that have critical neuronal functions. In support of this concept, it has been recently reported that Tip60 HAT activity exhibits neuroprotective functions in a Drosophila model for AD by repressing AD linked pro-apoptotic genes while loss of Tip60 HAT activity exacerbates AD linked neurodegeneration (Pirooznia, 2012a). However, whether misregulation of Tip60 HAT activity directly disrupts selective neuronal processes that are also affected by APP in vivo and the nature of such processes remains to be elucidated (Pirooznia, 2012 and references therein)

In Drosophila, the small and large ventrolateral neurons (henceforth referred to as sLNv and lLNv, respectively) are part of the well characterized fly circadian circuitry. Recent studied have implicated the l-and s-LNvs as part of the 'core' sleep circuitry in the fly, an effect that is predominantly coordinated via the neuropeptide pigment dispersing factor (PDF) that serves as the clock output, mediating coordination of downstream neurons. PDF is thought to be the fly equivalent of the mammalian neurotransmitter orexin/hypocretin because of its role in promoting wakefulness and thus stabilizing sleep-wake cycles in the fly. Within this circuit, the sLNvs are a key subset of clock neurons that exhibit a simple and stereotypical axonal pattern that allows high resolution studies of axonal phenotypes using specific expression of an axonally transported reporter gene controlled by the Pdf-Gal4 driver or by immunostaining for the Pdf neuropeptide that is distributed throughout the sLNv axons. These features make the sLNvs an excellent and highly characterized model neural circuit to study as they are amenable to cell type specific manipulation of gene activity to gain molecular insight into factors and mechanisms involved in CNS axonal regeneration as well as those that mediate behavioral outputs like sleep-wake cycle. Importantly, the Drosophila ventrolateral neurons (LNvs) have been previously used as a well characterized axonal growth model system to demonstrate that the AD linked amyloid precursor protein (APP) functions in mediating the axonal arborization outgrowth pattern of the sLNv. Based on these results, and previous studies reporting that Tip60 HAT activity itself is required for neural function and mediates APP induced lethality and CNS neurodegeneration in an AD fly model , It is hypothesized that APP and Tip60 are both required to mediate selective neuronal processes such as sLNv morphology and function that when misregulated, are linked to AD pathology. In the present study, this hypothesis was tested by utilizing the sLNvs as a model system to examine whether Tip60 mediated epigenetic dysregulation under neurodegenerative conditions such as that induced by APP overexpression leads to axonal outgrowth defects and if there is a corresponding effect on sLNv function in sleep regulation, a process that is also affected in neurodegenerative diseases like AD (Pirooznia, 2012).

This report shows that Tip60 is endogenously expressed in both the sLNv and lLNvs. Specific loss of Tip60 or its HAT activity causes reduction of PDF expression selectively in the sLNvs and not the lLNv and shortening of the sLNv distal synaptic arbors which are essential for the pre-synaptic release of PDF from these cells. The functional consequence of these effects is evidenced by the disruption of the normal sleep-wake cycle in these flies, possibly through disruption of PDF mediated signaling to downstream neurons. By using transgenic fly lines that co-express full length APP or APP lacking the Tip60 interacting C-terminus with a dominant negative HAT defective version of Tip60, it was demonstrated that the APP C-terminus enhances the susceptibility of the sLNvs and exacerbates the deleterious effects that the loss of Tip60 HAT activity has on axon outgrowth and PDF expression. Importantly, these studies identify the neuropeptide PDF as a novel target of Tip60 and APP, that when misregulated results in sleep disturbances reminiscent to those observed in AD. Remarkably, overexpression of wild type Tip60 with APP rescues these sleep defects by increasing PDF expression and inducing overelaboration of the sLNv synaptic arbor area. Taken together, these findings support a neuroprotective role for Tip60 on sLNv growth and function under APP induced neurodegenerative conditions. The data also reveal a novel mechanism for PDF control via Tip60 and APP that provide insight into understanding aspects of sleep dependent mechanisms that contribute to early pathophysiology of AD (Pirooznia, 2012).

Selective vulnerability of specific neuronal populations to degeneration even before disease symptoms are seen is a characteristic feature of many neurodegenerative diseases. Consistent with these studies, this study shows that when induction of the dTip60 RNAi response or expression of the dTip60 HAT mutant was directed to both the small and large LNvs, only the sLNvs were susceptible to the mutant effects induced under these conditions while the lLNvs were spared. The lack of any morphological effect on the lLNvs in the dTip60^E431Q flies could stem from the fact that compared to the sLNvs, these neurons express higher levels of endogenous Tip60 that counteracts the mutant dTip60^E431Q protein. However, induction of the RNAi response causes complete loss of Tip60 expression in both the lLNv and sLNv, and yet only the sLNvs are affected while the lLNv are spared, similar to the findings with dTip60^E431Q expression. This suggests that the sLNvs may be more susceptible to misregulation of Tip60 or its HAT activity. Of note, the dTip60^WT flies did not have any marked effect on the lLNv either, likely because these neurons are not susceptible to the moderate increase in Tip60 levels in the lLNvs induced under these conditions compared to the sLNvs. Developmentally, the sLNvs are known to differentiate much earlier than the large cells and this developmental difference may also in part account for the selective vulnerability of the sLNvs. In many neurodegenerative diseases, axon degeneration is known to involve protracted gradual 'dying-back' of distal synapses and axons that can precede neuron cell body loss and contribute to the disease symptoms. Importantly, loss of synapses and dying back of axons are also considered as early events in brain degeneration in AD. While APP overexpression in the LNvs did not have any observable effect on the sLNv axon growth at normal physiological temperatures, coexpression of the dTip60 HAT mutant with APP C-terminus appears to cause the sLNv axons in the adult animals to retract. The lack of any effect on the sLNv axon in the third instar larva in this case indicates that the axons grow to their full potential in the larval stage, but undergo degeneration post-mitotically in a process similar to 'dying-back' (Pirooznia, 2012).

A functional interaction between Tip60 and the amyloid precursor protein (APP) intracellular domain (AICD) has been shown to epigenetically regulate genes essential for neurogenesis. Such an effect is thought to be mediated by recruitment of the Tip60/AICD containing complex to certain gene promoters in the nervous system that are then epigenetically modified by Tip60 via site specific acetylation and accordingly activated or repressed. While the ^E431Q mutation in dominant negative HAT defective version of Tip60 (dTip60^E431Q) reduces Tip60 HAT activity, it should not interfere with its ability to assemble into a protein complex. Thus, dTip60^E431Q likely exerts its dominant negative action over endogenous wild-type Tip60 via competition with the endogenous wild-type Tip60 protein for access to the Tip60/AICD complex and/or additional Tip60 complexes, with subsequent negative consequences on chromatin histone acetylation and gene regulation critical for nervous system function. This study shows that co-expression of HAT defective Tip60 (dTip60^E431Q) with APP in the APP; dTip60^E431Q flies exacerbates the mutant effects that either of these interacting partners has on the sLNv axon growth and Pdf expression when expressed alone. In contrast, co-expression of additional dTip60^WT with APP alleviates these effects and this rescue is dependent upon the presence of the AICD region of APP. Thus, Tip60 HAT activity appears to display a neuroprotective effect on axonal outgrowth, Pdf expression, with concomitant alleviation of sleep defects under APP expressing neurodegenerative conditions. It is proposed that Tip60 might exert this neuroprotective function either by itself or by complexing with other peptides such as AICD for its recruitment and site specific acetylation of specific neuronal gene promoters to redirect their expression and function in selective neuronal processes such as sLNv morphology and function. Such a neuroprotective role for Tip60 is consistent with previous work demonstrating that excess dTip60^WT production under APP expressing neurodegenerative conditions in the fly rescues APP induced lethality and CNS neurodegeneration and that dTip60 regulation of genes linked to AD is altered in the presence of excess APP (Pirooznia, 2012). It is speculated that the degenerative effects observed in the APP; dTip60^E431Q flies may result from formation of Tip60^E431Q/AICD complexes that ultimately cause activation or de-repression of factors that promote axonal degeneration while excess Tip60/AICD complex formation in the APP;dTip60^WT expressing flies promote gene regulation conducive for sLNv outgrowth and Pdf expression (Pirooznia, 2012).

Sleep or wake promoting neurons in the hypothalamus or brainstem are known to undergo degeneration in a number of neurodegenerative diseases resulting in sleep dysregulation. In AD, such sleep disturbances are characterized by excessive daytime sleepiness and disruption of sleep during the night. These features resemble the symptoms of narcolepsy, a sleep disorder caused by general loss of the neurotransmitter hypocretin/orexin. Hypocretin is involved in consolidation of both nocturnal sleep and diurnal wake and loss of hypocretin levels have been correlated with sleep disturbances observed in AD. While the neuropathological changes in AD may contribute to hypocretin disturbances, a direct and causative role for APP in regulating hypocretin expression is not yet known. The LNv specific neuropeptide PDF is postulated to be the fly equivalent of hypocretin and has been shown to promote wakefulness in the fly. Consistent with these reports, the current data demonstrating somnolence during the light phase due to knock-down of PDF in the sLNv further supports a wake-promoting role for PDF. Accordingly, it was observed that overexpression of APP in the LNvs results in reduction of sLNv PDF expression as well as sleep disturbances that intriguingly, have been associated with AD pathology. The presence of similar effects on PDF and sleep due to loss of dTip60 HAT activity supports a role for both APP and Tip60 in controlling the PDF mediated sleep-wake regulation pathway. Previous studies have reported that the circadian modulators CLOCK and CYCLE regulate PDF expression in the sLNvs but not in the lLNvs. This study also observed a similar sLNv specific regulation of PDF by dTip60 in the adult flies. However, there was no effect on PDF expression in sLNvs in the larvae when Tip60 levels are undetectable. This is also consistent with the sLNv axonal defects that persist only in the adult flies. This suggests that the sLNvs may be subject to differential regulation during development as well as a temporal requirement for Tip60 in these cells in the adult flies. A recent study reported persistence of morning anticipation and morning startle response in LD in the absence of functional sLNv that were ablated due to expression of the pathogenic Huntington protein with poly glutamine repeats (Q128). Consistent with the Sheeba study, this study did not observe any marked effect on the morning and evening anticipatory behavior in LD in the dTip60^E431Q flies that exhibit a partial reduction in sLNv PDF. However, while the Q128 expressing flies were arrhythmic under constant darkness, dTip60^E431Q flies maintain rhythmicity in DD indicating that the sLNvs are still functional in these flies. The remarkable cell specificity of PDF regulation indicates the presence of additional as yet unidentified clock relevant elements or developmental events that distinguish between the two cell types (Pirooznia, 2012).

Recent evidence indicates that LNvs are light responsive and that their activation promotes arousal through release of PDF. Furthermore, PDF signaling to PDF receptor (PDFR) expressing neurons outside the clock, such as those found in the ellipsoid body that directly control activity, is thought to be important in translating such arousal signals into wakefulness. Since PDF is released from the sLNv axon terminals, the retraction of the sLNv axon terminals induced by the Tip60 HAT mutant can interfere with PDF mediated interaction of the sLNvs with downstream circuits. In the case of APP overexpression, while sLNv axon structure is unaffected, PDF expression is reduced; it is speculated that the decrease in PDF under these conditions is responsible for the abnormal sleep phenotype observed. In support of this theory, it was found that expression of APP lacking the C-terminus that also has no observable effect on the sLNv axon growth or PDF expression did not have any effect on sleep behavior. Thus the results indicate that the degenerative effect on the sLNv axons and/or the effect on PDF expression could both contribute to the observed sleep disturbances. Likewise, co-expression of the dTip60 HAT mutant with full length APP or APP lacking the C-terminus affected both the sLNv axon growth and PDF expression and consequently resulted in similar sleep disturbances (Pirooznia, 2012).

In addition to the wake promoting role, the LNvs also express GABAA receptors and are thus subject to inhibition by sleep promoting GABAergic inputs, analogous to those from the mammalian basal forebrain that regulate hypocretin neurons. The current consensus view is that sleep regulation is mediated by mutually inhibitory interactions between sleep and arousal promoting centers in the brain. The normal release of PDF from LNvs is part of the arousal circuitry in the fly and determines the duration of the morning and evening activity peaks while inhibition of these neurons and thus reduction in PDF release is necessary for normal sleep. Current models of sleep regulation suggest that the drive to sleep has two components, the first component is driven by the circadian clock and the second component is homeostatic in nature and the strength of this drive is based upon the amount of time previously awake. PDF release from sLNvs axon terminals exhibits diurnal variation and its release increases the probability of wakefulness by activating arousal promoting centers. However, the homeostatic drive for sleep that accumulates during the wake period eventually inhibits such arousal centers to promote sleep. Consistent with these reports, the reduction of PDF observed due to either dTip60^E431Q expression alone or co-expression of dTip60^E431Q with APP that leads to flies sleeping more during the day may also lead to a decrease in their homoeostatic drive for sleep, thus resulting in the less consolidated sleep patterns observed for these flies during the night. Conversely, it was found that overexpression of sLNv PDF due to dTip60 overexpression induces wakefulness and arousal. Additionally, these flies exhibit impaired ability to maintain sleep at night that may be mediated through inappropriate activation of arousal circuits due to PDF overexpression. Similar effects have been reported in a Zebrafish model due to hypocretin overexpression that results in hyperarousal and dramatic reduction in ability to initiate and maintain a sleep-like state at night. Despite the moderate increase in sLNv PDF levels in the dTip60^Rescue flies, no marked effect on sleep-wake cycle was observed in these flies. Extracellular levels of PDF and its signaling at synapses is thought to be regulated by neuropeptidases like neprilysin (see Neprilysin4). In fact, neprilysin mediated cleavage of PDF has been shown to generate metabolites that have greatly reduced receptor mediated signaling. Thus, it is speculated that the lack of any corresponding effect on sleep in the Tip60Rescue flies could be because such small increases in PDF might be regulated by endopeptidases like neprilysin. Based on these studies, a model is proposed by which the overelaborated sLNv synaptic arbors observed in flies co-expressing Tip60^WT and APP may provide additional input sites for signals from sleep promoting neurons in the vicinity that counteract the arousing effect of PDF overexpression on nocturnal sleep (Pirooznia, 2012).

Light mediated release of PDF from the lLNvs has been reported to modulate arousal and wakeful behavior as well as sleep stability. Thus, it has been suggested that the lLNvs may be part of an arousal circuit that is physiologically activated by light and borders with, but is distinct from the sLNvs and downstream sleep circuits. However, other studies have suggested that both LNv sub-groups promote wakeful behavior and that the lLNv act upstream of the sLNv. The observation of sLNv directed effects on PDF expression and the persistence of sleep-wake disturbances suggest that the sLNvs may be part of the neural circuitry that regulates sleep downstream of the lLNvs via a PDF dependent mechanism. In this regard, the sLNvs may participate in the communication between the lLNvs and other brain regions to promote light mediated arousal. It has been proposed by that the lLNvs may promote neural activity of the Ellipsoid body (EB) in the central complex (CC), a higher center for locomotor behavior that expresses the PDF receptor. However, disruption of sleep-wake cycles was observed even in the absence of any marked effect on the lLNv morphology or PDF expression. While no direct projections from the lLNvs to the EB have been detected, the sLNv axonal projections are relatively closer to the CC and thus may promote PDF receptor mediated signaling in such regions that control activity. Sleep disturbances, while prominent in many neurodegenerative diseases are also thought to further exacerbate the effects of a fundamental process leading to neurodegeneration. For these reasons, optimization of sleep-wake pattern could help alleviate the disease symptoms and slow the disease progression. In this regard, the modulatory effects that Tip60 HAT activity (dTip60^E431Q versus dTip60^WT) has on the sLNvs, the fly counterpart of the mammalian pacemaker cells, under APP overexpressing conditions, may provide novel mechanistic insights into epigenetic regulation of neural circuits that underlie behavioral symptoms like the 'sundowners syndrome' in AD. Future investigation into the downstream mechanism by which Tip60 regulates the sleep-wake cycle may further provide insight into the utility of specific HAT activators as therapeutic strategies for sleep disturbances observed in AD (Pirooznia, 2012).

Allatostatin A signalling in Drosophila regulates feeding and sleep and is modulated by PDF

Feeding and sleep are fundamental behaviours with significant interconnections and cross-modulations. The circadian system and peptidergic signals are important components of this modulation, but still little is known about the mechanisms and networks by which they interact to regulate feeding and sleep. This study shows that specific thermogenetic activation of peptidergic Allatostatin A (AstA)-expressing posterior lateral protocerebrum (PLP) neurons and enteroendocrine cells reduces feeding and promotes sleep in the fruit fly Drosophila. The effects of AstA cell activation are mediated by AstA peptides with receptors homolog to galanin receptors subserving similar and apparently conserved functions in vertebrates. The PLP neurons are identified to be a downstream target of the neuropeptide pigment-dispersing factor (PDF), an output factor of the circadian clock. PLP neurons are contacted by PDF-expressing clock neurons, and express a functional PDF receptor demonstrated by cAMP imaging. Silencing of AstA signalling and continuous input to AstA cells by tethered PDF changes the sleep/activity ratio in opposite directions but does not affect rhythmicity. Taken together, these results suggest that pleiotropic AstA signalling by a distinct neuronal and enteroendocrine AstA cell subset adapts the fly to a digestive energy-saving state which can be modulated by PDF (Chen, 2016).

Neuropeptides and peptide hormones transfer a wide variety of neuronal or physiological information from one cell to the other by activating specific receptors on their target cells. Most if not all peptides are pleiotropic and can orchestrate diverse physiological, neuronal or behavioural processes. In vertebrates, such a pleiotropic effect is especially prominent in the regulation of feeding and sleep. Many different peptides (e.g. orexin/hypocretin, ghrelin, obestatin) modulate different aspects of both behaviours, which reciprocally influence each other. The temporal pattern of neuroendocrine activity and neuropeptide release is shaped by sleep homeostasis and the circadian clock which, in turn, reciprocally affects feeding and sleep-wake cycles. Significant progress has been made in this field during recent years. Still little characterised, however, is the neuronal architecture that enables the relevant peptidergic neurons to integrate energy status, circadian time and sleep-wake status in order to coordinate the timing of sleep, locomotor activity and feeding. Information about the output signals by which endogenous clocks provide time- and non-circadian information to relevant peptidergic cells is still limited (Chen, 2016).

During the last years, the fruit fly Drosophila has become an important model for research into the regulation of feeding and sleep. Drosophila offers advanced genetic tools, a small brain with only about 100.000 neurons and a quantifiable sleep- and feeding behaviour that shows characteristics very similar to that of mammals. These features greatly facilitate the analysis of the neuronal and endocrine underpinnings of feeding and sleep. Like in most animals, feeding and sleep follow a circadian pattern in the fruit fly with little characterised neuronal and hormonal pathways downstream of the central clock. Like in mammals, a number of neuropeptides have been shown to be involved in the regulation of feeding or sleep in Drosophila. Yet, so far, only sNPF and likely also NPF are implicated in the regulation of both feeding and sleep. Also Insulin-like peptide (DILP)-expressing neurons (IPCs) in the pars intercerebralis affect feeding and sleep, yet only feeding seems to be directly dependent on DILP signalling (Chen, 2016).

Recent work by Hergarden (2012) demonstrated that neurons expressing neuropeptides of the allatostatin A (AstA) family regulate feeding behaviour of the fruit fly. Constitutive activation of AstA cells contained in the AstA¹-Gal4 expression pattern by ectopic expression of the bacterial low threshold voltage-gated NaChBac channel potently inhibited starvation-induced feeding. In contrast, constitutive inactivation of AstA¹ cells by expression of the inwardly rectifying Kir2.1 potassium channel increased feeding under restricted food availability. NaChBac activation of AstA¹ cells also inhibited the starvation-induced increase of the proboscis extension reflex (PER), a behavioural indicator for glucose responsiveness (Hergarden, 2012). The AstA¹ expression pattern includes a large number of brain neurons plus gut-innervating thoracico-abdominal ganglion (TAG) neurons and enteroendocrine cells (EECs) in the posterior midgut (Hergarden, 2012). This broad expression pattern is consistent with earlier described patterns of AstA-like immunoreactivity and suggests multiple functions for AstA. Earlier work had demonstrated an effect of AstA on gut motility. Two AstA receptors, DAR-1 (= AlstR) and DAR-2 are characterised for Drosophila. Different genome-based phylogenetic GPCR analyses independently demonstrated their homology with the galanin receptor family of vertebrates (Chen, 2016).

Using anatomical subdivision and genetic manipulation of neuronal activity, this study aimed to identify AstA functions and assign them to subsets of AstA expressing cells. The results revealed new interconnected AstA functions that link feeding and sleep and identify AstA-expressing PLP neurons and EECs as a target of the central clock output factor PDF. Pleiotropic AstA signalling seems capable of coordinating multiple aspects of physiology and behaviour in a coherent manner to adapt the fly to a digestive energy-saving state. The functional range of AstA signalling in the fly is thus reminiscent of the pleiotropy found in mammalian galanin signalling (Chen, 2016).

This study shows that AstA cells via AstA signalling subserve an anorexigenic and sleep-promoting function in Drosophila. In mammals, a variety of neuropeptides and peptide hormones affect both sleep and feeding, and the results provide evidence that also further such peptides exist in the fly besides sNPF and possibly NPF. More specifically, the results with a new AstA³⁴-Gal4 driver line show that activation of AstA-expressing PLP brain neurons or numerous EECs in the midgut strongly reduces food intake and promotes sleep. These behavioural effects are congruent with the anatomy of these cells. PLP interneurons are well positioned to modulate sleep as they widely arborise in the posterior superior protocerebrum, a projection area of sleep-relevant dopaminergic neurons, superior (dorsal) fan-shaped body neurons and neurons of the pars intercerebralis. AstA EECs in Drosophila are 'open type' EECs, possessing apical extensions that reach the gut lumen and likely express gustatory receptors. AstA-expressing EECs are thus potentially able to humorally signal nutritional information from the gut to brain centres regulating feeding and possibly also sleep and locomotor activity. If AstA is involved in inhibiting feeding and promoting sleep, one could expect AstA mutants to display decreased sleep and increased feeding in the absence of any other manipulation of AstA cells. It was observed, however, that a functional loss of the AstA gene did neither affect feeding nor locomotor activity under the experimental conditions with unrestricted access to a food source. This may suggest that AstA signalling is not part of a core feeding network, but represents an extrinsic modulator which becomes activated under specific yet so far uncharacterised conditions. Alternatively, as suggested by the observed difference in effect of constitutive vs. conditional electrical silencing of AstA cells, flies may be able to genetically or neuronally compensate for a constitutive loss of AstA signalling during development (Chen, 2016).

In larval Drosophila, AstA inhibits midgut peristalsis and affects K⁺ transport in order to concentrate ingested food. Together with the finding of a sleep-promoting and feeding-inhibiting effect of AstA, it is proposed that pleiotropic AstA signalling serves to coordinate behaviour and gut physiology to allow for efficient digestion. After food intake, AstA from the PLP neurons or EECs cause inhibition of further feeding, and -as the need for food search behaviour is relieved and nutrients need to be taken up- promotes sleep and inhibits gut peristalsis. Based on the gut content, enteroendocrine AstA is released and hormonally activates DAR-2 on key metabolic centers to tune adipokinetic hormone and insulin signalling, and -at least in other insects- stimulates digestive enzyme activity in the midgut (Chen, 2016 and references therein).

The AstA receptors are homologues of the vertebrate galanin receptors that have pleiotropic functions. When activated in specific brain areas, galanin signalling has a strong orexigenic effect and has also been implicated in the control of arousal and sleep in mammals. In zebrafish, transgenic heat-shock induced expression of galanin decreased swimming activity, the latency to rest at night and decreased the responsiveness to various stimuli. Furthermore, the allatostatin/galanin-like receptor NPR-9 inhibits local search behaviour on food in the nematode C. elegans. Similar to AstA in Drosophila, galanin modulates intestinal motility and ion transport. Thus, in broad terms, the involvement of DARs/galanin receptors in modulating feeding, gut physiology and arousal/sleep appears to be evolutionarily conserved (Chen, 2016 and references therein).

The neuronal clock network in Drosophila is intrinsically and extrinsically modulated by a variety of peptides (sNPF, NPF, calcitonin-gene related peptide/DH31, ion transport peptide, myoinhibiting peptides and PDF), which all affect sleep and locomotor activity and in part also act as clock output factors. Imaging results and constitutive activation of the PDF signalling pathway by t-PDF now suggest that the PLP neurons are modulated by PDF originating from the sLNv clock neurons. Unlike the peptides above, AstA from PLP neurons is outside and downstream of the central clock and seems not to modulate the clock network. Due to their anatomy and position, PLP neurons thus appear well-suited candidate cells by which clock neurons could modulate the complex cross-regulatory network regulating sleep, locomotor activity and perhaps also feeding. The rather mild effects on sleep and feeding of either t-PDF expression in AstA cells or thermogenetic activation of the sLNvs implies that this pathway is not the major output target of the central clock (if there is any) to modulate feeding and locomotor activity/sleep. This study found no shift in the circadian period or phase of feeding and locomotory activity/sleep upon AstA cell activation, suggesting that the main function of PDF-to-AstA cell signalling is not to time the respective behaviours but to modulate their amplitude. Similar non-timing functions of PDF have been demonstrated for other behaviours, including geotaxis and rival-induced mating duration (Chen, 2016).

At first sight, the current data suggesting that PDF activates PLP neurons to promote sleep seem to contradict earlier findings. Since pdf⁰¹ mutants show increased sleep during the photophase, the arousal effect appears to be the dominant effect of PDF which is due to signalling between ventral lateral clock neurons (LNvs), with a major contribution of the PDF-expressing large LNvs. The PLP neurons are only contacted by the sLNvs, which upon activation induced a time-specific increase in sleep, but did not increase arousal. Thus, the sLNv-PLP pathway likely represents a sleep-promoting clock output branch. Besides PDF, the sLNvs but not the lLNvs also co-localise the sleep-promoting peptide sNPF. A recent report shows that hormonal PDF released from abdominal PDF neurons serves to couple the central clock with a peripheral clock in the oenocytes. Furthermore, the posterior midgut is innervated by the abdominal PDF neurons, and PDFR is expressed in the midgut. It is thus possible that the AstA-expressing EECs represent additional PDF targets and may contribute to the PDF-related effects of AstA cells (Chen, 2016).

In conclusion, the lack of effect on feeding upon AstA cell silencing under non-restricted food availability and an unaltered circadian locomotor rhythmicity after AstA cell silencing suggests that AstA signalling is neither a primary signal in feeding regulation nor in the clock output pathway timing rhythmic behaviour. Rather-like mammalian galanin signalling - it seems to be one out of several modulatory pathways that allow to adapt the intensity of feeding and locomotor activity/sleep to specific physiological or environmental conditions. For example, decreased locomotor activity to save energy and increased digestion efficiency to maximise energy uptake may be most important during restricted food conditions, at which AstA cell silencing leads to increased feeding (Hergarden, 2012). While our results allow now to raise such speculations, it is clear that more research is needed to reveal the conditions at which AstA signalling is functional and the modulatory PDF input is strongest (Chen, 2016).

Aberrant axonal arborization of PDF neurons induced by Aβ42-mediated JNK activation underlies sleep disturbance in an Alzheimer's model

Impaired sleep patterns are common symptoms of Alzheimer's disease (AD). Cellular mechanisms underlying sleep disturbance in AD remain largely unknown. In this study, using a Drosophila Aβ42 AD model, Aβ42 was shown to markedly decrease sleep in a large population, accompanied with postdevelopmental axonal arborization of wake-promoting pigment-dispersing factor (PDF) neurons. The arborization is mediated in part via JNK activation and can be reversed by decreasing JNK signaling activity. Axonal arborization and impaired sleep are correlated in Aβ42 and JNK kinase hemipterous mutant flies. Image reconstruction revealed that these aberrant fibers preferentially project to pars intercerebralis (PI), a fly brain region analogous to the mammalian hypothalamus. Moreover, PDF signaling in PI neurons was found to modulate sleep/wake activities, suggesting that excessive release of PDF by these aberrant fibers may lead to the impaired sleep in Aβ42 flies. Finally, inhibition of JNK activation in Aβ42 flies restores nighttime sleep loss, decreases Aβ42 accumulation, and attenuates neurodegeneration. These data provide a new mechanism by which sleep disturbance could be induced by Aβ42 burden, a key initiator of a complex pathogenic cascade in AD (Song, 2016).

Fluorescence circadian imaging reveals a PDF-dependent transcriptional regulation of the Drosophila molecular clock

Circadian locomotor behaviour is controlled by a pacemaker circuit composed of clock-containing neurons. To interrogate the mechanistic relationship between the molecular clockwork and network communication critical to the operation of the Drosophila circadian pacemaker circuit, new fluorescent circadian reporters were established that permit single-cell recording of transcriptional and post-transcriptional rhythms in brain explants and cultured neurons. Live-imaging experiments combined with pharmacological and genetic manipulations demonstrate that the neuropeptide pigment-dispersing factor (PDF) amplifies the molecular rhythms via time-of-day- and activity-dependent upregulation of transcription from E-box-containing clock gene promoters within key pacemaker neurons. The effect of PDF on clock gene transcription and the known role of PDF in enhancing PER/TIM stability occur via independent pathways downstream of the PDF receptor, the former through a cAMP-independent mechanism and the latter through a cAMP-PKA dependent mechanism. These results confirm and extend the mechanistic understanding of the role of PDF in controlling the synchrony of the pacemaker neurons. More broadly, the results establish the utility of the new live-imaging tools for the study of molecular-neural interactions important for the operation of the circadian pacemaker circuit (Sabado, 2017).

A series of suppressive signals within the Drosophila circadian neural circuit generates sequential daily outputs

The Drosophila circadian neural circuit was studied using whole-brain imaging in vivo. Five major groups of pacemaker neurons display synchronized molecular clocks, yet each exhibits a distinct phase of daily Ca2+ activation. Light and neuropeptide pigment dispersing factor (PDF) from morning cells (s-LNv) together delay the phase of the evening (LNd) group by approximately 12 hr; PDF alone delays the phase of the DN3 group by approximately 17 hr. Neuropeptide sNPF, released from s-LNv and LNd pacemakers, produces Ca2+ activation in the DN1 group late in the night. The circuit also features negative feedback by PDF to truncate the s-LNv Ca2+ wave and terminate PDF release. Both PDF and sNPF suppress basal Ca2+ levels in target pacemakers with long durations by cell-autonomous actions. Thus, light and neuropeptides act dynamically at distinct hubs of the circuit to produce multiple suppressive events that create the proper tempo and sequence of circadian pacemaker neuronal activities (Liang, 2017).

Oscillating PDF in termini of circadian pacemaker neurons and synchronous molecular clocks in downstream neurons are not sufficient for sustenance of activity rhythms in constant darkness

In Drosophila, neuropeptide Pigment Dispersing Factor (PDF) is expressed in small and large ventral Lateral Neurons (sLNv and lLNv), among which sLNv are critical for activity rhythms in constant darkness. Studies show that this is mediated by rhythmic accumulation and likely secretion of PDF from sLNv dorsal projections, which in turn synchronises molecular oscillations in downstream circadian neurons. Using targeted expression of a neurodegenerative protein Huntingtin in LNv, a selective loss is evoked of neuropeptide PDF and clock protein Period from sLNv soma. However, PDF is not lost from sLNv dorsal projections and lLNv. These flies are behaviourally arrhythmic in constant darkness despite persistence of PDF oscillations in sLNv dorsal projections and synchronous Period oscillations in downstream circadian neurons. PDF oscillations in sLNv dorsal projections are not sufficient for sustenance of activity rhythms in constant darkness and this is suggestive of an additional component that is possibly dependent on sLNv molecular clock and PDF in sLNv soma. Additionally, despite loss of Period in sLNv, their activity rhythms entrain to light/dark cycles indicating that sLNv molecular clocks are not necessary for entrainment. Under constant light, these flies lack PDF from both soma and dorsal projections of sLNv, and when subjected to light/dark cycles, show morning and evening anticipation and accurately phased morning and evening peaks. Thus, under light/dark cycles, PDF in sLNv is not necessary for morning anticipation (Prakash, 2017).

Protein Interactions

PDF receptor signaling in Drosophila contributes to both circadian and geotactic behaviors

The neuropeptide Pigment-Dispersing Factor (PDF) is a principle transmitter regulating circadian locomotor rhythms in Drosophila. A Class II (secretin-related) G protein-coupled receptor (GPCR) has been identified that is specifically responsive to PDF and also to calcitonin-like peptides and to PACAP. In response to PDF, the PDF receptor (PDFR) elevates cAMP levels when expressed in HEK293 cells. As predicted by in vivo studies, cotransfection of Neurofibromatosis Factor 1 significantly improves coupling of PDFR to adenylate cyclase. pdfr mutant flies display increased circadian arrhythmicity, and also display altered geotaxis that is epistatic to that of pdf mutants. PDFR immunosignals are expressed by diverse neurons, but only by a small subset of circadian pacemakers. These data establish the first synapse within the Drosophila circadian neural circuit and underscore the importance of Class II peptide GPCR signaling in circadian neural systems (Mertens, 2005).

An antiserum against the final 20 amino acids of the predicted C terminus of PDFR was used to establish sites of PDFR expression within the adult brain. In the wild-type adult brain, the most prominent PDFR immunosignals revealed a large cell body in the dorsal-lateral protocerebrum. Roughly 20 neuronal cell bodies were stained in the anterior and medial subesophageal ganglion (SEG). In addition, scores of more weakly stained soma were detected in all regions of the brain, especially along the superficial aspects of the medulla. Strong staining of neuronal processes was evident throughout the central brain and optic lobes. These immunosignals were lost when the antibody was preincubated with the immunizing peptide, but were not altered in either the pdfr P1 or P2-36 mutant stocks (Mertens, 2005).

Throughout the brain, PDF-positive processes were always associated with PDFR-positive processes. For example, the projection of the small LNv neurons was closely associated with PDFR-positive puncta in the dorsal protocerebral neuropil. Single optical sections revealed that the PDF-positive terminals were closely apposed to PDFR-labeled processes. PDF-positive projections within the median bundle were likewise in proximity to abundant PDFR-positive processes. The large PDF-expressing LNv neurons make a broad tangential projection along the distal medulla. Notably, numerous PDFR-stained cells and processes were evident in areas of the medulla and lobula that lacked PDF-stained processes. Along the lateral aspect of the medulla, little evidence was seen of receptor processes immediately adjacent to the tangential PDF projection except in the anterior aspect (Mertens, 2005).

It was asked whether any circadian pacemaker neurons (as assayed through Per immunostaining) coexpressed PDFR. Receptor expression was found in only a subset of defined circadian pacemaker neurons. A prominent pair of DN1 neurons was positive for both Per and for PDFR; these two cells were closely abutted to the dorsal surface of the brain and placed anterior to the other DN1s. Two to three DN3 neurons were weakly PDFR immunopositive. The DN2 neurons, large LNv neurons, LNd neurons and small LNv neurons all lacked PDFR immunosignals. PDFR immunosignals did not vary diurnally. pdfr mRNA did not exhibit diurnal or circadian variation according to results of RNA profiling; instead, it was regulated at a steady-state level by per (Mertens, 2005).

Evidence indicates that in Drosophila PDF signals via a Class II GPCR that is most closely related to the calcitonin-CGRP receptor family. The EC_[50] of ~25 nM measured in this study is likely an overestimate that reflects the heterologous expression system that was employed. PDFR signaling properties in vitro parallel published accounts of PDF actions in vivo. PDF elicits increases in cAMP in vivo, and the PDFR appears coupled to G_s in HEK293 cells. Also, genetic analysis showed that the NF1 protein operates downstream of PDF to support circadian output (Williams, 2001). Similarly, this study found that cotransfection of dNF1 in HEK293 cells greatly increases the efficacy of PDF in producing high-amplitude signaling through the PDFR. This effect is reminiscent of NF1 coupling another Class II peptide GPCR, the PACAP receptor (PAC 1), to adenylate cyclase (Dasgupta, 2003). While Class II peptide GPCRs typically couple to G_s, many Class II receptors also signal via calcium, including CGRP receptors and VPAC receptors). Similarly, PDFR signaling also increased calcium levels, albeit with a much higher EC_[50] value in comparison with the effect on cAMP levels. In all, these data provide a basis for future evaluation of PDF receptor properties in situ (Mertens, 2005).

While PDF-related peptide and DNA sequences appear to be restricted to invertebrate lineages, its receptor (CG13758) is clearly related to certain mammalian receptors (Brody, 2000). These observations suggest that there is conservation of the PDF signaling pathway between arthropods and chordates. PDFR is a Class II peptide GPCR, and other members of this category (e.g., PACAP and VIP receptors) exert profound influences in the mammalian circadian system. In some respects, the functional roles of PDF in the fly circadian system and VIP in the mouse are parallel. Both peptides are required for the normal display of behavioral rhythms in constant conditions -- producing short period rhythms or arrhythmicity -- and both affect the rhythmicity of cellular pacemaking (VIP). Among the mammalian Class II GPCRs, PDFR is more closely related to calcitonin and calcitonin-gene related peptide (CGRP) receptors than to either PACAP or VIP receptors. CGRP immunosignals and CGRP binding sites have been measured in the SCN. Nevertheless, the functional analogies of PDFR-like signaling to CGRP-R-like signaling may be limited, since receptor component protein (RCP), a protein that modulates CGRP responsiveness in a variety of cell types, does not affect PDFR coupling in the current experiments. It is notable that both Drosophila PDFR and mammalian VPAC receptors respond to PACAP peptides. In fact, PDFR was activated by PDF and PACAP-38, also by the peptides calcitonin, adrenomedullin, and a Drosophila ortholog of calcitonin called DH₃₁. Among these, PDF is clearly the most potent ligand and produces the strongest secondary signals. DH₃₁ activates a separate Drosophila Class II GPCR called CG17415, a receptor that is not sensitive to PDF. It is proposed that the PDF receptor displays partial agonism by diverse ligands, which is a common feature among Class II peptide GPCRs. For example, VPAC receptors demonstrate high-affinity interactions with VIP and PACAP and, to lesser extents, with other naturally occurring peptides such as GRF and secretin. PACAP-38 has several physiological effects in Drosophila tissues. Whether PDFR also represents an endogenous PACAP receptor in vivo is now open to investigation (Mertens, 2005).

To what extent can the properties of PDFR explain the in vivo behavioral signaling controlled by PDF? Four of the five Drosophila Class II GPCRs were tested and it was found that CG13758 alone displays sensitivity to PDF. The one untested Class II GPCR is CG12370, and, on the basis of its strong sequence similarity to CG8422, it likely encodes a CRF receptor-related receptor that is sensitive to the peptide DH₄₄. The genetic analysis to date does not allow exclusion of the contribution of other (potential) PDFRs to the regulation of circadian rhythmic behavior. However, the results clearly indicate that PDFR is primarily responsible for PDF signaling underlying the modulation of the Drosophila geotactic behavioral response. Two results underscore this point. pdfr alleles produced a geotactic phenotype as severe as that displayed by pdf mutant flies. Also, flies transheterozygous for pdf and either of two distinct pdfr mutations displayed a strong mutant phenotype, while individual heterozygotyes were not distinguished from controls. Together, these data clearly link the actions of pdf and pdfr within the same physiological pathway. The simplest hypothesis to explain these results is that PDFR is the primary receptor for PDF in the context of geotactic behavior. Why do certain alleles of pdfr display a strong geotactic phenotype, but not a strong locomotor phenotype? Those results are consistent with published properties of the pdf mutant flies indicating that the geotaxis assay is sensitive to small increments of PDF signaling (Toma, 2002). It is proposed that such sensitivity may underlie the differential effects that was measured with receptor mutants. It follows that definition of the complete locomotor phenotype of pdfr mutant flies awaits recovery of stronger mutant alleles. Indeed, this prediction is fully met by analysis of a naturally-occurring mutation of the pdfr (CG13758) locus that produces a circadian locomotor defect which closely matches that of pdf (Lear, 2005). That independent genetic data strongly supports the contention that CG13758 encodes the principle PDF receptor in Drosophila (Mertens, 2005).

In general, excellent correspondence was found between PDFR-positive processes and PDF-positive processes in diverse brain regions. In the dorsal brain, the trajectory and extent of processes from the small LNv neurons are closely matched by receptor-positive processes. Likewise, receptor processes are closely intermingled with PDF-positive varicosities in the anterior medulla and the median bundle. By contrast, many receptor-positive processes were tens of microns away from the closest PDF-positive processes. These distances do not necessarily preclude physiological interactions between PDF and PDFR, as indicated by studies of 'receptor mismatches'. These and other examples support the concept of volume transmission, which refers to the diffusion of bioactive substances across considerable distances via the extracellular space. Given these antecedents and based on the proximity of receptor immunosignals to PDF signals in several areas, it is proposed that the pattern of PDFR expression is consistent with a role in mediating PDF signaling throughout the brain and optic lobes. An additional and nonexclusive hypothesis is that PDFR displays high-affinity interactions with more than one ligand (analogous to mammalian VPAC receptors), and this possibility is supported by its partial agonism, as was observed in vitro (Mertens, 2005).

PDF is a synchronizing factor that sustains or delays molecular oscillations within pacemaker neurons, including oscillations within the pacemakers that release PDF. However, the degree to which PDF acts directly or indirectly on pacemaker cells remains uncertain. The results of studying PDFR-like immunoreactivity do not support the hypothesis of broad, direct PDF action on pacemaker neurons. Among the ~150 brain pacemakers, PDFR immunosignals are only expressed by a pair of DN1s and more weakly by two to three scattered DN3s. The antibody could detect PDFR in most pacemaker cells when the protein was overexpressed, but not in native tissue. These results argue that, normally, most pacemaker neurons contain very low amounts of the receptor. Hence, it is suggested that the predominant influence of PDF on the synchronization of circadian pacemaker neurons proceeds via indirect neuronal connections. Specifically, these results focus attention on the prominent pair of PDFR-positive DN1 cells as potentially critical relay neurons within the circadian pacemaker network (Mertens, 2005).

In summary, the identification of a PDF receptor provides the basis for addressing PDF functions in a cellular context. Its sites of expression define potential sites of PDF actions. Its signaling properties will illuminate the mechanisms by which PDF modifies geotactic behavior and helps organize daily locomotor rhythms. (Mertens, 2005).

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

The neuropeptide Pigment-Dispersing Factor (PDF) plays a critical role in mediating circadian control of behavior in Drosophila. Mutants have been discovered in groom-of-PDF (gop) that display phase-advanced evening activity and poor free-running rhythmicity, phenocopying pdf mutants. In gop mutants, a spontaneous retrotransposon disrupts a coding exon of a G protein-coupled receptor, CG13758. Disruption of the receptor is accompanied by phase-advanced oscillations of the core clock protein Period. Moreover, effects on circadian timing induced by perturbation of PDF neurons require gop. Yet PDF oscillations themselves remain robust in gop mutants, suggesting that GOP acts downstream of PDF. gop is expressed most strongly in the dorsal brain in regions that lie in proximity to PDF-containing nerve terminals. Taken together, these studies implicate GOP as a PDF receptor in Drosophila (Lear, 2005).

In light:dark cycles, both pdf and gop mutants display reduced or absent anticipation of the morning “lights on” transition and a phase advance of the evening activity peak. In addition, both mutants display free-running rhythms in constant darkness that decay over time. Both mutants also exhibit phase-advanced molecular rhythms after several days in constant darkness. The striking similarity between gop and pdf mutants strongly suggests that these two genes operate in a discrete pathway. Interestingly, both pdf and gop mutants were discovered as spontaneous mutations (Lear, 2005).

Abundant evidence is available that the gene mutated in the gop mutant is the peptide GPCR CG13758. Complementation testing maps the gop phenotype away from other mutations on its X chromosome and to an area of 3A region containing just six candidate genes including CG13758. CG13758 transcripts are severely disrupted by a retrotransposon insertion in the third exon corresponding to a portion of the N-terminal extracellular domain. None of the mutant cDNAs analyzed would produce a wild-type full-length receptor. These data strongly suggest that the disruption of CG13758 is largely responsible for the gop phenotype (Lear, 2005).

Tenetic and phenotypic analysis suggests that GOP acts downstream of PDF: (1) PDF remains rhythmically expressed in gop mutants; (2) alteration of circadian period through manipulation of clock genes in PDF⁺ neurons is blocked in gop mutants, indicating that gop is required for PDF neuronal effects on circadian phase. Although essential for PDF neuron action, the gop transcript is most strongly expressed in cells thought to be the targets of PDF neurons in the dorsal brain, likely including the pars intercerebralis and perhaps the DN1s. Indeed, changes are observed in Per expression in a subset of DN1s in the dorsal brain. Finally, preliminary data is available that the extracellular domain of GOP can bind PDF using an in vitro assay. Moreover, this binding appears to be specific. It is competed by PDF but not by another neuropeptide, proctolin, and significant binding is not observed with an unrelated peptide. Further studies using broader sets of ligands and quantitative affinity assessments in combination with cell-based signaling studies will be necessary to definitively identify GOP as the PDF receptor. The behavioral phenotype described in this study suggests that GOP may be the sole PDF receptor in Drosophila (Lear, 2005).

The gop expression pattern is also consistent with a role as a PDF receptor. gop expression was noted most prominently in the dorsal brain. There is a wealth of evidence that PDF release into the dorsal brain mediates circadian behavior. This study found gop transcript expression in parts of the dorsal brain in the regions of the LNv terminals, including areas near the pacemaker DN1 neurons as well as the pars intercerebralis. The idea is favored that these sites of receptor expression mediate circadian behavior. While these cells cannot be definitely identified as the DN1 group, it is interesting to note that Per expression in a subset of DN1s is altered in gop mutants, consistent with potential GOP function in these neurons. Nonetheless, cell-specific double-labeling experiments will be necessary to demonstrate clearly gop expression in DN1 pacemaker neurons (Lear, 2005).

These studies also clearly reveal a role for GOP in feedback onto the core oscillator. Previous studies described binding of biotinylated PDF directly on subsets of LNs and DNs. No significant gop expression is observed in the LNs yet molecular oscillations in the LNs are altered. The notion is favored that the effects of gop on LN oscillations are not cell-autonomous, but instead reflect altered network function. Anatomic studies have defined a potentially reciprocal circuit between LNvs and DN1s. Such a circuit may be necessary to reinforce molecular cycling under constant conditions. It is proposed that altered signaling in the DN1s due to loss of gop may modulate feedback onto the LNvs and result in advanced Per cycling in these cells. Consistent with this hypothesis reduced Per staining was observed in a subset of DN1s in gop mutants (Lear, 2005).

CG13758/gop encodes a class B GPCR neuropeptide receptor most closely related to calcitonin receptors (Brody, 2000). Of note, rhythmic calcitonin receptor expression has been observed in the mammalian circadian pacemaker, the suprachiasmatic nucleus. More compelling is the apparent role of peptidergic class B GPCR signaling in both fly and mammalian circadian clocks. Like gop mutants, genetic knockout of the VPAC2 receptor, a class B GPCR that is equally activated by VIP and pituitary adenylate cyclase-activating peptide (PACAP), results in a loss or alteration of behavioral and molecular rhythms (Harmar, 2002). Thus, these studies reveal underlying similarities in clock mechanisms beyond the core transcriptional feedback loops. The identification of a G protein-coupled receptor essential for PDF neuronal action represents an important step toward elucidating the molecular and neural pathways that transform core molecular oscillations into daily behavioral rhythms (Lear, 2005).

Drosophila GPCR Han is a receptor for the circadian clock neuropeptide PDF

The pigment-dispersing factor (PDF) is a neuropeptide controlling circadian behavioral rhythms in Drosophila, but its receptor is not yet known. From a large-scale temperature preference behavior screen in Drosophila, a P insertion mutant was isolated that preferred different temperatures during the day and night. This mutation, which was named han, reduces the transcript level of CG13758. Han was expressed specifically in 13 pairs of circadian clock neurons in the adult brain. han null flies showed arrhythmic circadian behavior in constant darkness. The behavioral characteristics of han null mutants are similar to those of pdf null mutants. It was also found that PDF binds specifically to S2 cells expressing Han, which results in the elevation of cAMP synthesis. Therefore, it is proposed that Han is a PDF receptor regulating circadian behavioral rhythm through coordination of activities of clock neurons (Hyun, 2005).

PDF is expressed in l-LNvs and s-LNvs and is secreted in the axon termini of these neurons. These neurons have large arborizations and send projections to contralateral and dorsal areas of the brain where LNd, DN1, DN2, and DN3 are present. In contrast, the PDF receptor Han is expressed in 13 pairs of neurons in adult fly brains: four l-LNvs, one LNd, seven DN1s, and one DN3. Therefore, it is reasonable to conclude that the PDF signals produced by l-LNvs and s-LNvs are transmitted to four l-LNvs, one LNd, seven DN1s, and one DN3 through Han receptors. LNv, LNd, DN1, and DN3 neurons play certain roles in the control of circadian rhythmic behaviors and, thus, PDF-Han signaling might also play some roles in coordinating interactions between these groups of clock neurons (Hyun, 2005).

Comparing expression patterns of PDF reveals, interestingly, that Han is expressed only in l-LNv but not in s-LNvs. Both l-LNvs and s-LNvs express PDF. It has been suggested that s-LNv neurons are the most important master neurons of clock neurons in flies because strong oscillations of Per and Tim proteins are continuously sustained over five days in s-LNvs in DD. Although neurite fibers of l-LNv and s-LNv are inter-connected, oscillation of Per and Tim proteins in l-LNv is not obvious a few days after the 'light-off' in contrast to s-LNv neurons. Han-mediated PDF signaling may contribute to the coordinated interaction of l-LNv with s-LNv neurons because Han is expressed in l-LNv neurons. These two groups of clock neurons have been shown to control morning oscillators while LNd neurons control evening oscillators (Stoleru, 2004; Grima, 2004). In both pdf and han mutants, the morning oscillators, as well as the evening oscillators, do not function properly. Flies cannot properly anticipate the time when light will be on or off. This implies that PDF-Han signaling in four l-LNv neurons and one LNd neuron, in which Han is expressed, is necessary for the proper function of the morning and evening oscillators (Hyun, 2005).

Han is homologous to the mammalian CT receptor and the VPAC₂ receptor. The mouse VPAC₂ receptor is known to be important for the maintenance of circadian rhythmic behavior and core clock gene expression in mice (Harmar, 2002). Both the CT receptor and the VPAC₂ receptor are expressed in the mammalian clock center SCN. Therefore, it is possible that a similar mechanism to coordinate interactions between clock neurons via PDF-Han signaling may also be present in the mammalian brain (Hyun, 2005).

Screening mutants showing abnormal thermal preference originally led to the isolation of han. han^X7867 prefers a colder temperature than normal only during the night. However, han³³⁶⁹ and han⁵³⁰⁴, which is null, did not show clear differences in temperature preference during day and night. Instead, they consistently preferred a temperature of 23.5°C, slightly colder than normal. It is not known yet if Han has a role in temperature sensation or thermoregulation in flies. Because the pdf⁰¹ mutant has been reported to show abnormal geotactic behavior (Toma, 2002), whether han mutants exhibit any defects in geotaxis was examined by means of a simple climbing assay. In this assay, no significant geotactic phenotypes were observed in han mutants. This observation does not exclude the possibility that some subtle geotactic phenotypes are present in han mutants, which might be detectable by more elaborate analysis with the sophisticated apparatus described by Toma (2002). Han expression in neurons other than clock neurons such as those in ventral nerve cords in the larval brain suggests that Han may play roles other than regulating circadian rhythmic behaviors. The possible roles of Han in other physiological or behavioral phenomena such as temperature preferences or sensation need to be investigated further in connection with circadian rhythms (Hyun, 2005).

Autoreceptor control of peptide/neurotransmitter core lease from PDF neurons determines allocation of circadian activity in Drosophila

Drosophila flies concentrate behavioral activity around dawn and dusk. This organization of daily activity is controlled by central circadian clock neurons, including the lateral-ventral pacemaker neurons (LN_vs) that secrete the neuropeptide PDF (pigment dispersing factor). Previous studies have demonstrated the requirement for PDF signaling to PDF receptor (PDFR)-expressing dorsal clock neurons in organizing circadian activity. Although LN_vs also express functional PDFR, the role of these autoreceptors has remained enigmatic. This study shows that (1) PDFR activation in LN_vs shifts the balance of circadian activity from evening to morning, similar to behavioral responses to summer-like environmental conditions, and (2) this shift is mediated by stimulation of the Gα,s-cAMP pathway and a consequent change in PDF/neurotransmitter co-release from the LN_vs. These results suggest another mechanism for environmental control of the allocation of circadian activity and provide new general insight into the role of neuropeptide autoreceptors in behavioral control circuits (Choi, 2012).

Studies of the Drosophila circadian control circuit over the past decade have revealed a critical role for neuropeptide signaling between PDF secreting LN_vs and PDF-negative PDFR-expressing clock neurons for generation and maintenance of robust circadian activity rhythms. While PDF-secreting LN_vs also express PDFR, little is known about the functional significance of these peptide autoreceptors, other than enhancing the robustness of free-running rhythms in DD. This study shows that PDFR autoreceptor activation in LN_vs shifts the balance of daily circadian activity to the morning from the evening by engaging the Gα,s-cAMP pathway and thereby modulating secretion of PDF and co-released classical synaptic neurotransmitter (Choi, 2012).

While PDFR expression can be detected in both the sLN_vs and lLN_vs, the current results suggest that PDFR-induced increased morningness is likely mediated by small LN_vs (sLN_vs) rather than large LN_vs (lLN_vs). First, sLN_vs respond robustly to bath-applied PDF with increased cAMP whereas lLN_vs do not, and downregulating Gα,s activity or cAMP levels suppresses the behavioral effects of LN_v PDFR activation. Second, PDFR autoreceptor activation in LN_vs cell-autonomously depolarizes the sLN_vs while not affecting the lLN_vs. Third, expression of t-PDF with the sLN_v-specific R6-GAL4 driver induces a more robust increase in morningness than with the lLN_v-specific c929-GAL4 driver. Nevertheless, these findings do not rule out some contribution of PDFR autoreceptors expressed in lLN_vs (Im, 2010) in the membrane-tethered PDF (t-PDF) induced shift of the balance of daily activity from evening to morning. While PDFR activation by bath-applied PDF does not induce a cAMP increase in lLN_vs, it is possible that lLN_v PDFR couples to non-cAMP signaling pathways such as Ca²⁺ signaling. However, the suppression of t-PDF induced increased morningness by disruption of Ga,s-cAMP signaling indicates that any contribution of lLN_v PDFR activation occurs upstream of the sLN_vs (Choi, 2012).

This study demonstrate that increased morningness induced by PDFR activation in LN_vs requires not only PDF outputs (as expected from prior studies) but also classical synaptic neurotransmitter co-release by the LN_vs. Other behaviors such as feeding also employ multiple co-released intercellular signals with different dynamics, with one signal instructing the behavior and the other modulating the sensitivity to the instructive signal (Root, 2011). There is also evidence that neuropeptide and inhibitory synaptic neurotransmitter co-secretion underlies unique behavioral adaptations. Determining the identity of the co-released classical neurotransmitter required for PDFR autoreceptor-mediated increased morningness will provide insight into the question how sLN_v outputs influence downstream circadian clock neurons to determine daily activity rhythms (Choi, 2012).

What is the physiological significance of the increased morningness induced by PDFR activation sLN_vs? One clue is that this PDFR autoreceptor-induced increased morningness mimics the behavioral response of flies to summer-like lighting conditions, where morning activity is increased and/or phase advanced. This suggests that PDF signals to LN_vs might be regulated by light conditions. Indeed, lLN_vs are highly sensitive to light, contact sLN_v post-synaptic sites, promote phase-advances to morning light, and their PDF secretion is important for entrainment by light (Cusumano, 2009). Thus, the lLN_vs are well-situated to transfer illuminance information to the sLN_vs via PDF to regulate allocation of circadian activity and phase as photoperiod changes throughout the year. Consistent with this hypothesis, hyperexcitation of lLN_vs by expression of NaChBac (low threshold bacterial voltage-gated Na²⁺ channel) increases circadian morning activity. This leads to the model of a homotypic PDF relay circuit, where summer conditions with lengthened and brighter dawn increase PDF secretion by lLN_vs, thus increasing PDFR activation in sLN_vs, thereby adaptively adjusting the allocation of circadian activity between morning and evening. Interestingly, a recent study has shown a strong temperature dependence of the timing of morning activity in natural light and temperature conditions and an induction of a prominent daytime activity bout at very high temperatures (Vanin, 2012), suggesting that temperature also regulates the distribution of daily activity. It is interesting to speculate that this temperature modulation of daily activity allocation relies on PDFR autoreceptors and the intra- and inter-celluar signaling pathways elucidated in this study (Choi, 2012).

There are many parallels between the model for PDFR autoreceptor signaling in the Drosophila circadian control network and VIP signaling in the mammalian circadian network of the SCN. They include the fact that VIP expressing cells in the SCN are innervated by the visual system and that VIP secretion is stimulated by light. Moreover, there is evidence that VIP signals modulate SCN transcriptional responses to light, mediate light-dependent phase-shifts of circadian locomotor rhythms. About 30% of all SCN VIP cells also express VIPR, suggesting that VIPR autoreceptor signaling in the SCN plays a similar role to that of PDFR in modulating VIP/classical neurotransmitter co-release Intriguingly, the possibility of mammalian VIPR autoreceptor signaling influencing light-dependent circadian behavior can be tested using the GPI-tethered peptide strategy employed in this study in the fly. VIPR is a class B1 G protein coupled receptor (GPCR), and previous studies have shown the GPI-tethered peptide design developed in the context of the fly is generalizable to mammalian class B1 GPCR ligands. Indeed, GPI-tethered VIP (t-VIP) was tested in vitro in heterologous cells and found to be a potent activator of VIPR. By expressing t-VIP using the VIP promoter, analogous to expression of t-PDF using the pdf promoter, one can test whether VIPR autoreceptor signaling modulates VIP-dependent circadian behaviors such as arousal and phase shifts. Further studies of this nature will elucidate the cellular mechanisms by which peptide autoreceptor signaling modulates sleep/wake and activity control circuits as well as reveal evolutionarily conserved principles for the modulation of daily behavioral rhythms by the environment (Choi, 2012).

DEVELOPMENTAL BIOLOGY

A single Pdf transcript (ca. 0.8 kb) is expressed predominantly in the head; the expression levels of PDF mRNA are consistently higher in males than in females (Park, 1998). There are no detectable hybridization signals in the head of the arrhythmic disconnected mutant, regardless of sex. This seems to contradict the immunocytochemical studies showing that, despite the absence of PDF immunoreactivity in lateral neurons anterior to the medulla optic lobe, 8-16 PDH-immunoreactive neurons near the mushroom-body calix in the dorsal brain are still intact in this mutant (Helfrich-Foster, 1997 and 1998). Therefore, it is suggested that the vast majority of the PDF mRNA is derived from PDF-containing lateral neurons. Alternatively, the dorsally located neurons may be nonspecifically immunostained due to the use of antibody against crustacean peptide. Clarification of this issue awaits the use of a specific antibody raised against genuine Drosophila PDF (Park, 1998).

Antisera against the crustacean pigment-dispersing hormone (beta-PDH) were used in immunocytochemical preparations to investigate the anatomy of PDH-immunoreactive neurons in the nervous system of wild-type Drosophila melanogaster and in that of several brain mutants of this species, some of which express altered circadian rhythmicity. In the wild-type and in all rhythmic mutants (small optic lobes, sine oculis, and small optic lobes;sine oculis) double mutants, eight cell bodies at the anterior base of the medulla (PDFMe neurons) exhibit intense PDH-like immunoreactivity. Four of the eight somata are large and four are small. The four large PDFMe neurons have wide tangential arborizations in the medulla and send axons via the posterior optic tract to the contralateral medulla. Fibers from the four small PDFMe neurons ramify in the median protocerebrum, dorsal to the calyces of the mushroom bodies. Their terminals are adjacent to other PDH-immunoreactive somata (PDFCa neurons) which send axons via the median bundle into the tritocerebrum. The results suggest a possible involvement of the PDFMe neurons in the circadian pacemaking system of Drosophila. The location and size of the PDFMe neurons are identical with those of neurons containing the Period protein, which is essential for circadian rhythmicity. Changes in the arborizations of the PDFMe neurons in small optic lobes;sine oculis double mutants are suited to explain the splitting in the locomotor rhythm of these flies. In the arrhythmic mutant, disconnected, the PDFMe neurons are absent. The arrhythmic mutant per0, however, shows normal PDH immunoreactivity and therefore, does not prevent the expression of PDH-like peptides in these neurons (Helfrich-Forster, 1993).

The Period protein (Per) is a essential component of the circadian clock in Drosophila. Although Per-containing pacemaker cells have been previously identified in the brain, the neuronal network that comprises the circadian clock remained unknown. Some Per plus neurons are also immunostained with an antiserum against the crustacean pigment-dispersing hormone (PDH). This antiserum reveals the entire arborization pattern of these pacemaker cells. The arborizations of these neurons are appropriate for modulation of the activity of many neurons, and they might interact with Per-containing glial cells (Helfrich-Foster, 1995).

In adults, most of the ventral group of LNs (LN_vs) are immunoreactive to the peptide hormone PDH. This PDH immunoreactivity in the group of smaller LN_vs (small LN_vs) persists from early larval stage through the metamorphosis. In the current study per-beta-gal fusion gene construct called BG expression persists in a similar set of LNs. If these BG-expressing LNs in larvae are PDH-immunoreactive, it would support the idea that LNs in larvae are indeed the precursors of a subset of LN_vs in the adults. Thus, L3 brains of BG were double-labeled for X-gal and anti-PDH at ZT 0. The double-labeling procedure was performed on 22 brains (44 brain hemispheres) and gave reliable results on 41brain hemispheres. The numbers of LNs labeled by either BG expression or PDH presence, or both were counted. In ~80% of the valid brain hemispheres, four or five LNs were positive for either BG or PDH, or both. In most of the samples in which five LNs were positive, all of the LNs were stained for BG, but the number of PDH-stained LNs never exceeded four. These PDH-stained LNs also were stained for BG in all 12 such cases. In most of the specimens for which four LNs were revealed, all four of the neurons were indeed double-labeled for PDH and BG. These results indicate that there are five larval LNs, of which four contain Per and PDH, suggesting that these four doubly expressing neurons correspond to the precursor of small LN_vs in adults, whereas one LN contains only Per and had not been identified previously in adults. One further feature of the PDH-related results was that the immunohistochemical signals revealed the projections of the four small LN_vs in the double-labeled preparations. Interestingly, their terminals were always located in close proximity to the cell bodies of dorsal neurons-2^Larval (DN2^Ls) (Kaneko, 1997).

Pigment-dispersing hormones (PDH) are a family of octadecapeptides that have been isolated from several crustacean species. An antiserum against the crustacean PDH was used to identify PDH-immunoreactive neurons in the developing nervous systems of wild type Drosophila and the brain mutant disconnected. Particular attention was paid to a group of PDH-immunoreactive neurons at the anterior margin of the medulla, known as the pigment-dispersing factor-containing neurons close to the medulla (PDFMe neurons). This group of neurons seems to be involved in the control of adult circadian rhythms. In adults, this group consists of four to six neurons with large somata (large PDFMe neurons) and four neurons with small somata (small PDFMe neurons). Both the small and the large PDFMe neurons are identical to the ventral lateral neurons, a group of neurons containing the Period protein. Both subgroups are usually absent in adults of behaviorally arrhythmic disconnected mutants. The compound eyes of these mutants are usually disconnected from the optic lobes due to a severe defect in optic lobe development. disco mutants, as a result, have either very tiny rudiments of optic lobes if no connections are made at all (unconnected phenotype) or, if some connections are established (connected phenotype), the optic lobes have an almost normal size but are grossly disorganized. disco mutants are behaviorally arrhythmic, and the lateral neurons are generally absent in adults. In the wild type, PDH immunoreactivity is seen first in the small PDFMe neurons of 4 hour old first-instar larvae. The small PDFMe neurons persist unchanged into adulthood, whereas the large ones seem to develop halfway through metamorphosis. In addition to the PDFMe neurons, three other clusters of PDH-immunoreactive neurons stain in the developing nervous systems of Drosophila and are described in detail. Two of them are located in the brain, and the third is located in the abdominal neuromeres of the thoracic nervous system. In the mutant disconnected, the larval and the adult set of PDFMe neurons are absent. The other clusters of PDH-immunoreactive neurons seemed to develop normally. The present results are consistent with the hypothesis that the PDFMe neurons are circadian pacemaker neurons that may control rhythmic processes in larvae, pupae, and adults (Helfrich-Forster, 1997).

Mutations at the disconnected (disco) locus of Drosophila disrupt neural cell patterning in the visual system, leading to the loss of many optic lobe neurons. Drosophila's presumptive circadian pacemaker neurons (the dorsal and ventral lateral neurons) are usually among the missing cells, and most disco flies are behaviorally arrhythmic. Ventral lateral neurons (LNvs) are occasionally present and provoke robust circadian rhythmicity in disco mutants. Of 357 individual disco flies four animals with robust circadian rhythmicity were found. All four retained LNvs together with terminals in the superior protocerebrum. Residual or bi-circadian rhythmicity was found in about 20% of all flies; the remaining flies were completely arrhythmic. One of the flies with residual rhythmicity and two of the arrhythmic flies also had some LNvs stained. However, these flies lacked the LNv fibers in the superior protocerebrum. The results suggest that the presence of single LNvs is sufficient to provoke robust circadian rhythmicity in locomotor activity if the LNv terminals reach the superior protocerebrum. The presence of residual or bi-circadian rhythmicity in 20% of the flies without LNvs indicates that other cells also contribute to the rhythmic control of locomotor activity (Helfrich-Forster, 1998).

beta-pigment-dispersing hormone (beta-PDH) isolated from the fiddler crab is a member of an octadecapeptide family of neuropeptides common to arthropods. Whereas earlier studies of these peptides in insects had been limited to orthopterans, this investigation focuses on dipteran flies. Extracts of heads from the blowfly Phormia terraenovae were assessed in a fiddler crab bioassay for PDH activity. Immunocytochemistry, dose-response curves, gel filtration chromatography and reversed-phase HPLC, combined with bioassay and enzyme-linked immunosorbent assay (ELISA), indicate the presence of PDH-like peptide in the blowfly. Immunocytochemical mapping of PDH-like immunoreactive (PDHLI) neurons with a beta-PDH antiserum was performed for the entire nervous systems of Phormia and the fruitfly Drosophila. In the cephalic ganglion (brain, optic lobe and subesophageal ganglion) PDHLI cell bodies could be detected (34 in Phormia and 16 in Drosophila). In both species, each hemisphere contains 8 PDHLI cell bodies in the optic lobes. These innervate the optic lobe neuropils bilaterally. In Phormia, another set of 8 cell bodies are located in each of the lateral neurosecretory cell groups in the superior protocerebrum. These neurons send axons to the corpora cardiaca-hypocerebral ganglion complex and to portions of the foregut. In contrast, only the optic lobe neurons display immunoreactivity in Drosophila. Except for the optic lobes, PDHLI processes are distributed only in nonglomerular neuropils of the brain in both species. In the fused thoracico-abdominal ganglia of Phormia, 28 PDHLI cell bodies are found (only six are found in Drosophila). In both species, six abdominal PDHLI neurons are efferents with axons innervating the hindgut. Some of the PDHLI neurons in the Phormia brain and abdominal ganglion contain colocalized FMRFamide-like immunoreactivity. Since the flies studied here do not display hormonally controlled, fast pigment migrations, the PDH-like peptide may have a role as neurotransmitter or neuromodulator in the central nervous system, especially in the visual system, and a regulatory role in the stomatogastric system and the hindgut (Nassel, 1993).

Axon caliber in monopolar cells L1 and L2 of the fly's lamina can change dynamically. Swelling by day, L2 exhibits a daily rhythm of size changes apparently mediated by wide-field LBO5HT and PDH cells. L1/L2 axon profiles were measured planimetrically in the housefly, Musca domestica, from 1 microns cross sections. Four hours after injecting 5-HT into the optic lobe, L1's axon swells but L2's does not, whereas PDH enlarges both axons. Similar to 5-HT, histamine (the photoreceptor transmitter) enlarges L1 but not L2, mimicking light exposure, while glutamate GABA and both decrease L1 and L2. 5,7-dihydroxytryptamine decreases L2 and, somewhat, L1, an effect attributable to the loss of LBO5HT neurites. Twenty four hours after cutting LBO5HT and PDH commissural pathways, L1 and L2 both shrink. Apparently, the size of L2 depends on either LBO5HT or sufficient 5-HT, and L1 and L2 have different response ranges to 5-HT. Responses to PDH imply that daytime PDH release drives a circadian rhythm, enlarging L1 and L2 (Pyza, 1996).

Circadian control of eclosion: Interaction between a central and peripheral clock

Drosophila displays overt circadian rhythms in rest:activity behavior and eclosion. These rhythms have an endogenous period of approximately 24 hr and can adjust or 'entrain' to environmental inputs such as light. Circadian rhythms depend upon a functioning molecular clock that includes the core clock genes period and timeless. Although a clock in the lateral neurons (LNs) of the brain controls rest:activity rhythms, the cellular basis of eclosion rhythms is less well understood. The LN clock has been shown to be insufficient to drive eclosion rhythms. The prothoracic gland (PG), a tissue required for fly development, contains a functional clock at the time of eclosion. This clock is required for normal eclosion rhythms. However, both the PG clock function and eclosion rhythms require the presence of LNs. In addition, it is demonstrated that pigment-dispersing factor (PDF), a neuropeptide secreted from LNs, is necessary for the PG clock and eclosion rhythms. Unlike other clocks in the fly periphery, the PG is similar to mammalian peripheral oscillators because it depends upon input, including PDF, from central pacemaker cells. This is the first report of a peripheral clock necessary for a circadian event (Myers, 2003).

Lateral neurons (LNs) are considered the central circadian pacemaker. These LNs are required for rest:activity rhythms and are most likely required for controlling the timing of eclosion (adult emergence from the pupal case). Eclosion is considered to be under the control of the circadian system because its timing is gated such that it is restricted to the hours surrounding dawn each day, even for flies that are developmentally ready hours earlier. Because eclosion occurs once in a single fly's lifetime, the multiple events that occur over several days within a population are considered a rhythm. This gating is absent in flies mutant for the clock gene period (per) or timeless (tim) and is also absent in disconnected flies that lack LNs (Myers, 2003).

Neuronal clocks (including those in the LNs) are sufficient to drive rest:activity rhythms, but perhaps not eclosion rhythms. It was of interest to determine whether these clocks would be sufficient for eclosion gating by using fly lines in which the molecular clock functions in neurons only. The gal4-UAS binary system was used to express Tim only in neurons by using the elav^c155-gal4 driver and a UAS-tim transgene in an arrhythmic tim null background. This manipulation does not rescue eclosion rhythms as it does locomotor rhythms in adults (Myers, 2003).

Another circadian mutant line, one that also displays rhythmic rest:activity behavior, was also arrhythmic for eclosion. This fly line, cry^b, is mutant for a circadian photoreceptor, Cryptochrome (Cry). Cry^b affects the sensitivity of the LN molecular clock to pulses of light but does not affect its endogenous rhythm. Emerging evidence now suggests that Cry is a central clock gene in peripheral clocks. Besides demonstrating that the LN clock is insufficient to drive eclosion rhythms, the eclosion data suggest that Cry may also be required within a relevant peripheral clock mechanism (Myers, 2003).

From these data, it is concluded that a LN molecular clock, which can drive rest:activity rhythms, is not sufficient to restore eclosion rhythms. A peripheral clock, then, is necessary to maintain eclosion rhythms, even in the presence of a functioning LN clock (Myers, 2003).

The prothoracic gland (PG), is part of an endocrine structure known as the ring gland. This structure surrounds the heart just anterior to the cardia and is present during all stages of life except adulthood. The PG secretes ecdysteroids, which when converted to the active form of 20-hydroxyecdysone bind to their nuclear hormone receptor (ecdysone receptor, EcR) and affect gene transcription. These alterations in gene expression cause tissue metamorphosis over the course of development. Levels of ecdysteroids peak at the beginning of larval and pupal stages but, during the two days just prior to eclosion, drop to nearly undetectable levels. In Manduca sexta (tobacco hornworm), this drop in ecdysteroid titer is necessary for eclosion to proceed normally (Myers, 2003).

Previous studies suggest that there is some circadian control over PG function. Notably, ecdysone titers cycle in a circadian fashion in Rhodnius prolixus. In Drosophila, Per is present and oscillates in central brain-PG cultures taken from white prepupae. It is not known, however, whether both Per and Tim oscillate in this tissue under free-running conditions (in constant darkness and temperature) immediately preceding eclosion. Presumably, these conditions should be met before one considers the PG a true clock tissue and an appropriate candidate clock tissue involved in the control of eclosion gating (Myers, 2003).

Clock function was assessed in the PG by quantitating Per and Tim levels over the course of the day in intact pupae. Both Per and Tim levels change over the course of the LD cycle, with a significant difference between the peak (late night) and the trough (late day) values. The peak of Per expression is slightly later when compared to Tim. Both expression profiles, though, match those seen in the LNs. Per and Tim still show significant differences in daily expression in constant darkness (DD), although the difference between the peak and trough values is, as in other fly tissues, smaller. Per expression in DD, although significantly different throughout the course of the day, does not match its LD profile. This effect is similar to the delay in Per degradation in DD versus LD seen in head extracts, although the delay is more pronounced within the PG (Myers, 2003).

It is concluded that there is a molecular clock inside cells of the PG at the time when pupae are developmentally ready for imminent eclosion. Because differences between peak and trough levels of Per are smaller, the profiles of daily Tim expression in DD were used to report PG clock function (or clock synchrony within the population) in subsequent experiments (Myers, 2003).

Does circadian output of the PG clock gate eclosion? Because lesions of the PG are lethal, the necessity of the PG clock was established by assaying eclosion rhythms in fly lines in which genetic manipulation had disrupted the clock inside the PG. To disrupt the molecular clock inside the PG, Tim (UAS-tim^2-1) was expressed at all times of day specifically in PG cells by using the Mai⁶⁰-gal4 driver. Expressing Tim in this manner disrupts eclosion rhythms. Peaks are present in the UAS-tim^2-1; Mai⁶⁰-gal4 eclosion profile, but a rhythm and gating are absent. In this line, adult locomotor behavior remains rhythmic (80.3% of adults had a significant circadian period to their rest:activity behavior in constant light. Consistent with a role for the PG in eclosion gating, it was found that there are no significant differences in the daily expression of Tim in the PG of the arrhythmic cry^b flies (Myers, 2003).

Although the clock inside the LNs is not sufficient for eclosion rhythms, the cells still appear to be required for eclosion gating. It is likely that the LNs could control PG clocks, much like the suprachiasmatic nucleus (SCN) of the hypothalamus is believed to drive peripheral clocks in mammals. Anatomical evidence does suggest that the LN axons (containing pigment-dispersing factor, PDF) indirectly innervate the PG (Myers, 2003).

To determine whether LNs are necessary, tests were carried out for the presence of both eclosion rhythms, and a PG clock in a fly line that lacked LN cells. A fly line was used in which LNs were ablated without lesioning many other neurons. This is a more focused disruption than that caused by the disconnected (disco) mutation. LN cells were ablated by driving a cell death gene, head-involution defective (hid), with pdf-gal4. These pdf-gal4 X UAS-hid flies are also arrhythmic for rest:activity behavior as adults. In the PG of pdf-gal4 × UAS-hid and in disco⁰¹ flies, there is no longer any significant difference in Tim expression over the course of the day. Although eclosion gating in the pdf-gal4 X UAS-hid line appears to be present during the first two days in constant darkness, rhythms do not persist. It is speculated that the flies emerging during the first two days of this assay are gated because their exposure to the entraining LD cycle persists until relatively late in development. This may result in limited and short-lived synchrony through unknown mechanisms. Clearly, though, LNs are required to maintain eclosion rhythms (Myers, 2003).

To determine whether a functioning molecular clock inside the LNs is necessary for their influence on the peripheral clock and on eclosion, the effect of disrupting this clock was examined. Tim (UAS-tim^3-1) was expressed in neurons of wild-type flies at all times of day by using an elav^c155-gal4 driver, all in a wild-type background. This perturbation of Tim expression is sufficient to disrupt locomotor rhythms in the adult fly. Although the molecular clock in the LNs is disrupted, there are still daily changes of Tim in the PG, and eclosion remains rhythmic (Myers, 2003).

It is possible that the role of the LNs is to provide, via PDF, a signal to the PG clock. In the LN axons that project to the dorsal brain, PDF expression cycles, with PDF release believed to occur during subjective night. The rhythmicity of PDF release and eclosion correlate well. For instance, in per⁰ and tim⁰ flies, PDF is no longer released in a rhythmic fashion from these dorsal, LN projections, and eclosion is arrhythmic as well. To determine whether PDF is part of the LN output pathway to the PG or involved in eclosion gating, both PG clock function and eclosion were assayed in flies with no functional PDF protein (pdf⁰¹). These flies are arrhythmic for locomotor behavior as well as for eclosion. In the PG, Tim levels were significantly different over the course of the day in the presence of an LD cycle, but neither Per in LD nor Tim in DD showed significant differences in their daily expression profile. These data indicate that the endogenous clock inside the PG cannot function (or entrain) without PDF in the fly. PDF overexpression and anatomical studies suggest that the LNs are the best candidates for a source of PDF relevant to eclosion behavior and the PG clock. PDF, though, is also expressed in a small subset of neurons in the central brain (LN_vs and two to four tritocerebral cells) and in four to six abdominal cells. In addition, there may be other inputs to the PG. For instance, the Tim cycling seen in LD conditions is most likely due to an acute light response, suggesting the presence of photic input to the PG. Supporting this hypothesis are data from R. proxilus, whose PG clock (its presence is inferred from cyclic release of ecdysteroids in culture) is also directly photosensitive (Myers, 2003 and references therein).

Thus, the PG molecular clock is under control of the central clock. This is unlike other Drosophila peripheral clocks, such as those in the antenna, which can operate autonomously. It is also unexpected when one considers data from studies in which PG clock function is directly or indirectly assayed in culture and determined to be independent of the central brain (or independent of any tetrodotoxin-dependent output from the brain, in the case of Drosophila). The PG clock, in fact, is more similar to peripheral clocks in the mammalian circadian system. In addition, it is important to note that edysteroid synthesis (and presumably PG clock function) in the cockroach Periplaneta americana also depends upon input from the central brain. Perhaps, then, the mechanisms controlling peripheral clock function are not the same in different tissues in the same insect or in the same tissue in different insects (Myers, 2003).

From these data, a model for the circadian gating of eclosion emerges. The LNs secrete PDF into the anterior protocerebrum, where it acts on neurons that innervate the PG. Appropriate regulation of PDF levels is critical. Just as the absence of PDF disrupts eclosion, so can excess levels of it in the dorsal brain. However, the mechanisms underlying this PDF overexpression phenotype are unknown (Myers, 2003).

This study introduces a new set of clock cells necessary for the regulation of eclosion rhythms. It is not known, however, whether the LN and PG clocks together are sufficient to control eclosion gating. The current hypothesis holds that, in the ventral nervous system of Drosophila, cells containing Crustacean Cardioactive Peptide (see Cardioacceleratory peptide) are the most likely sites for control of eclosion gating, as indicated by two lines of evidence. The first is that CCAP, in response to eclosion hormone (EH), can activate ecdysis within minutes in Manduca sexta. However, the circadian gate of eclosion cannot be regulated solely by EH or CCAP because flies without either of these sets of neurons still eclose within a circadian gate. The second line of evidence is that some CCAP cells in Drosophila also express LARK, an RNA binding protein that regulates eclosion rhythms. LARK oscillates in a clock-dependent manner inside these cells. Interestingly, PG cells also contain LARK, although there have been no reports of cycling LARK outside of the CCAP cells. The exact mechanism for how and where development and circadian inputs are coordinated to control eclosion gating are still important and open questions (Myers, 2003 and references therein).

Functional analysis of circadian pacemaker neurons in Drosophila melanogaster

The molecular mechanisms of circadian rhythms are well known, but how multiple clocks within one organism generate a structured rhythmic output remains a mystery. Many animals show bimodal activity rhythms with morning (M) and evening (E) activity bouts. One long-standing model assumes that two mutually coupled oscillators underlie these bouts and show different sensitivities to light. Three groups of lateral neurons (LN) and three groups of dorsal neurons govern behavioral rhythmicity of Drosophila. Recent data suggest that two groups of the LN (the ventral subset of the small LN cells and the dorsal subset of LN cells) are plausible candidates for the M and E oscillator, respectively. Evidence is provided that these neuronal groups respond differently to light and can be completely desynchronized from one another by constant light, leading to two activity components that free-run with different periods. As expected, a long-period component starts from the E activity bout. However, a short-period component originates not exclusively from the morning peak but more prominently from the evening peak. This reveals an interesting deviation from the original Pittendrigh and Daan (1976) model and suggests that a subgroup of the ventral subset of the small LN acts as 'main' oscillator controlling M and E activity bouts in Drosophila (Rieger, 2006).

Daily biological rhythms are governed by inherent timekeeping mechanisms, called circadian clocks. Such clocks reside in discrete sites of the brain and consist of multiple autonomous single-cell oscillators. Within each neuron, interacting transcriptional and translational molecular feedback loops as well as ionic signaling pathways constitute the oscillatory mechanism of the clock. It is not understood how individual pacemaker neurons interact to drive behavioral rhythmicity. The long-standing model of Pittendrigh and Daan (1976) assumes that the clock consists of two groups of oscillators with different responsiveness to light, one governing the morning (M) and the other the evening (E) activity of the animal. Typical M and E activity bouts are present in animals ranging from insects to mammals and suggest that the two-oscillatory model is generally valid. It has been shown that M and E bouts could be eliminated or reinstated by manipulating different circadian pacemaker neurons in Drosophila. This work has suggested that the ventral (LN_v) and dorsal (LN_d) subsets of the lateral neurons are the neuronal substrates for the M and E oscillators. It is not known whether these two oscillators respond differently to light (Rieger, 2006).

The particular power of the two-oscillator model is that it explains observed adaptations to seasonal changes in day length. The model predicts that the M oscillator will shorten and the E oscillator will lengthen its period when exposed to extended constant light (LL). As a consequence, the M activity occurs earlier and the E activity occurs later in long summer days, helping day-active animals avoid the midday heat. The model also predicts that the M oscillator will free-run with short period and the E oscillator with long period when animals are placed in constant light. However, such internal desynchronization between oscillators does not occur, because high-intensity constant light usually results in arrhythmicity. In Drosophila, the clock protein Timeless (TIM) is permanently degraded during light-induced interaction with Cryptochrome (CRY), leading finally to the arrest of the clock. Without functional CRY, this does not happen. Indeed, internal desynchronization into two free-running components (one with a short period and the other with a long period) has been described for cry^b mutants under constant-light conditions. The present study aims to analyze the molecular state of all clock gene-expressing neurons during behavioral rhythm dissociation to test the Pittendrigh–Daan model and refine the neuronal substrates of the E and M oscillators (Rieger, 2006).

This study supports the notion that the activity rhythm of Drosophila is controlled by at least two sets of neuronal oscillators. Furthermore, the definition of these neuronal substrates of both oscillators were refined more precisely than previously. As proposed by Pittendrigh and Daan (1976), the two oscillators show different responses to light: one is accelerated and the other decelerated by constant light. However, a deviation from the original model was observed. In contrast to previous observations, the current results suggest that the PDF-positive s-LN_v cells control not only the M but also the E activity bout. Therefore, the discussion should perhaps not focus of a 'morning' oscillator but rather of an M–E or 'main' oscillator (to keep the 'M'), for the following reasons. The PDF-positive s-LN_v cells are essential for maintaining activity rhythms after several days under constant conditions, and electrical silencing of the LN_v cells severely impairs free-running rhythms. In the present study, the PDF-positive s-LN_v cells appear to dominate the rhythms in those flies that did couple E and M components after the first crossing-over on day 11 in LL, because such flies free-ran with short period (Rieger, 2006).

The hypothesis that the PDF-positive LN_v cells control not only the M activity but also partly the E activity can also explain other findings. The E activity bout is always the most prominent peak, which persists under constant-dark conditions, whereas the M activity bout is much reduced under such conditions and may even disappear. Thus, mainly the E component constitutes the free-running rhythm, and it seems implausible that the neurons responsible for rhythmicity under these conditions should have no impact on the E component. Indeed, it has been found that the s-LN_v show the most robust cycling after extended time under constant conditions. Furthermore, another study emphasizes the importance of the s-LN_v cells for the timing of activity peaks under constant conditions (Rieger, 2006 and references therein).

Despite their dominance, the PDF-positive s-LN_v cells depend on functional LN_d and DN cells to provoke a normal E activity bout under light-dark conditions. Flies with the clock gene PER present only in PDF-positive LN_v cells have a prominent M activity bout but lack the E activity bout. It is unclear whether this is attributable to the E activity fusing with the M activity or whether the E activity is suppressed, but these findings show that the output from the PDF cells requires PER in the LN_d and DN cells to manifest wild-type activity patterns (Rieger, 2006).

It was found that the PDF-negative 5th s-LN_v cell cycles in-phase with the LN_d cells under LL and thus may contribute to the E component. Notably, the PDF-negative 5th s-LN_v cell shows high-amplitude cycling. Although this is not proof of the involvement of this cell, it suggests that it is an important circadian pacemaker neuron. Little is known about this cell because it could not be distinguished from the other lateral neurons in the former studies in which single-labeled clock protein staining was performed, but the PDF-negative 5th s-LN_v cell is the only clock cell beside the PDF-positive s-LN_v cells that appears to work from the first larval instar onward. Thus, it might have the same strong impact on the activity rhythm that has been revealed for the PDF-positive s-LN_v cells. More work is necessary to reveal the role of the PDF-negative s-LN_v cell in more detail (Rieger, 2006).

Additional studies are also necessary to fully reveal the function of the DN cells. The current results suggest that the DN₁ and the DN₃ cells may contain different subclusters. Indeed, the DN₁ cells develop at different times and appear to have distinct projection patterns. It is very likely that some DN₁ cells contribute to the M oscillator whereas others supply the E oscillator. Again, there are data that support this hypothesis: if the lateral neurons (s-LN_v, l-LN_v, and LN_d) are absent as a result of mutation or genetic ablation but the dorsal neurons (DN₁, DN₂, and DN₃) are left intact, morning and evening activity bouts are still present under LD conditions, although with reduced amplitude and changed phase. The DN₂ cells might play a special role for bimodal activity patterns because, in wild-type flies, they cycle 12 h out-of-phase with the s-LN_v and LN_d cells under DD conditions. The present study indicates that this is not the case in cry^b flies under LL conditions, because the DN₂ cells were in-phase with all other neurons on the first day in LL. The same applies for wild-type flies under LD conditions. It has been shown that the DN₂ are indeed pacemaker neurons that cycle independently of the s-LN_v cells. However, despite their autonomous function, the DN₂ cells did not visibly contribute to the activity patterns of the flies under constant darkness. This suggests a minor role of the DN₂ cells in the control of the activity rhythm, but the possiblity cannot be exclude that the DN₂, together with the other DN groups, may contribute to morning and evening activity bouts under certain conditions (Rieger, 2006).

The blue-light photopigment cryptochrome is regarded as the main photoreceptor of the fruit flies' circadian clock. This study shows that the compound eyes are responsible for period shortening and period lengthening of the molecular oscillations in different subsets of pacemaker neurons (the M and E oscillators) under LL. Their special role may lie in the adaptation of the clock to seasonal changes. This is in line with previous findings showing that the compound eyes are necessary for the adequate timing of M and E activity bouts in long summer days and short winter days. Cryptochrome, conversely, appears to lengthen the period in all clock neurons as can be deduced from the periods of the wild-type flies that showed internal desynchronization under 'moonlight LL.' In such flies, the periods of both components were clearly longer than those of internally desynchronized cry^b flies (Rieger, 2006).

The internal desynchronization of activity into long- and short-period components described in this study is reminiscent of previous results for Drosophila mutants with primarily reduced optic lobes or ectopic expression of PDF. Both of these fly strains have ectopic PDF-containing nerve fibers in the dorsal brain that might lead to elevated and/or nonrhythmic secretion of PDF in this brain area and may disturb normal communication between the pacemaker cells. It is unknown whether such a perturbed communication results in internal desynchronization between the s-LN_v and the 5th s-LN_v and extra LN_d as observed in the present study. Dual-oscillator systems have been also described for mammals, but in no case they could be traced to the level of single neurons. Like the circadian pacemaker center of flies, the mammalian pacemaker center, the suprachiasmatic nucleus (SCN), contains a heterogeneous neuronal population. A recent study has shown that internal desynchronization of motor activity into short and long periods similar to the one shown in this study can be provoked in rats by special light schedules. As in Drosophila, both components reflect the separate activities of two oscillators in anatomically defined subdivisions of the SCN. Furthermore, there is some evidence to suggest that the SCN is composed of two oscillating M and E components. These results underline the universality of dual-oscillator systems (Rieger, 2006).

Other studies strongly implicate the PDF-expressing LN_v and the LN_d cells as the respective neuronal loci for the morning and evening activity bouts. Despite the near 12 h phase difference between the morning and evening locomotor peaks under LD, no obvious molecular phase differences between these pacemakers have been observed that would explain them. Work in mammals suggests that the relationship between molecular phase and locomotion is complex. For example, nocturnal and diurnal rodents show the same phases of PER oscillations. Furthermore, different rat strains that displayed unimodal or multimodal activity patterns, respectively, all exhibited the same unimodal rhythm in melatonin synthesis. Individual Nile grass rats changed their activity patterns from unimodal–diurnal to bimodal–nocturnal after introducing a running wheel. Despite this dramatic effect on the activity patterns, the wheel had little effect on the circadian pacemaker, and the spatial and temporal patterns of c-Fos expression in the SCN remained similar. All of these data indicate that the relationship between molecular and behavioral phase is not straightforward. Clearly, a multitude of phase relationships between the molecular rhythm and behavior are possible. Brain regions outside the pacemaker center may be responsible for these different phases as was shown recently for mammals. It appears that the same is true within the circadian system of the fly. The present data show that, during the internally synchronized state, the trough in PER level of all neurons correlates with the main activity bout (the E peak). No second trough appears to correlate with the M peak. However, a second small peak can be seen at closer inspection of the PDF immunoreactivity in the terminals of the s-LN_v. This suggests that the unimodal rhythm in clock protein cycling might be converted into a bimodal output already within the neurons (Rieger, 2006).

During the state of behavioral desynchronization under LL conditions, an internal desynchronization was observed simultaneously in PER oscillations among subsets of pacemaker neurons. One interpretation of these data is that constant light causes internal desynchronization between these pacemaker neurons that then in turn drive the behavioral outputs. However, it must be acknowledged that this is only a correlation, and, although the hypothesis is favored that the split molecular rhythms are driving the split locomotor rhythms, it is possible that they are merely tracking or entraining to a split rhythm driven by other pacemakers. For example, the split rhythms might be driven by subsets of dorsal neurons. The hypothesis is preferred that the split behavioral rhythms were driven by the desynchronized PDF-positive LN_v and the 5th s-LN_v/extra LN_d cells for two reasons. First, accumulating evidence points to the lateral neurons (LN_v and LN_d cells) as major pacemaker cells, whereas the dorsal neurons (the DN₁, DN₂, and DN₃ cells) are not sufficient for locomotor rhythms under constant darkness. Second, in rodents, a similar behavioral desynchronization was correlated with a dissociation of clock gene expression between ventrolateral and dorsomedial subdivisions of the SCN. The established role of this brain center as the circadian clock has led to the uncontroversial conclusion that the split molecular oscillations drive the split behavioral oscillations. It is suggested that the same phenomenon is occurring in main (i.e., small LN_v cells) and evening (i.e., 5th s-LN_v and extra LN_d cells) neuronal oscillators in Drosophila (Rieger, 2006).

A subset of dorsal neurons modulates circadian behavior and light responses in Drosophila

A fundamental property of circadian rhythms is their ability to persist under constant conditions. In Drosophila, the ventral Lateral Neurons (LNvs) are the pacemaker neurons driving circadian behavior under constant darkness. Wild-type flies are arrhythmic under constant illumination, but flies defective for the circadian photoreceptor CRY remain rhythmic. Flies overexpressing the pacemaker gene per or the morgue gene, a gene that can protect flies from the disruptive effects of constant light when overexpressed with the tim-GAL4 driver, are also behaviorally rhythmic under constant light. Unexpectedly, the LNvs do not drive these rhythms: they are molecularly arrhythmic, and PDF - the neuropeptide they secrete to synchronize behavioral rhythms under constant darkness - is dispensable for rhythmicity in constant light. Molecular circadian rhythms are found only in a group of Dorsal Neurons: the DN1s. Thus, a subset of Dorsal Neurons shares with the LNvs the ability to function as pacemakers for circadian behavior, and its importance is promoted by light (Murad, 2007).

Recent studies have shown that two groups of cells control circadian behavior. The PDF-positive LNvs are called morning cells (M cells), and the LNds evening cells (E cells), because they control the anticipatory behavior observed before dawn and dusk, respectively. In addition, the LNvs are the cells maintaining circadian behavior in constant darkness and controlling the phase of most circadian neurons of the brain. In their absence, circadian behavior rhythms are lost after a few days in DD. Surprisingly, the current results show that a functional circadian clock in the LNvs is actually not necessary for long-term behavioral rhythms. In flies overexpressing PER, the LNvs are no longer circadianly functional under constant illumination. No oscillation of the circadian protein PDP1 can be detected, and yet these flies remain rhythmic for at least 7 days. Moreover, limiting per overexpression to circadian neurons that do not express PDF is sufficient to obtain circadian behavioral rhythms under constant environmental conditions (Murad, 2007).

It is thought that the neurons maintaining circadian behavior independently of the LNvs are not the E cells. Indeed, when per is overexpressed, no sign of circadian oscillation is seen in the neurons that are thought to control the evening activity: the LNds. In addition, the PDF-negative LNv that might also contribute to the evening activity did not cycle in LL when morgue was overexpressed. Moreover, flies with per overexpression driven by cry-GAL4 were completely arrhythmic under constant light. cry-GAL4 is one of the critical GAL4 drivers used to define the E cells. Importantly, molecular circadian oscillations were detected in only one group of cells when per was overexpressed: the DN1s. Due to the high number of DN3s, it cannot be ruled out that a few cells in the DN3 groups also oscillate. Interestingly, it has been shown that a subset of DN3 neurons can maintain their own circadian oscillations in DD, in the absence of circadianly functional LNvs. However, these DN3 cells were not able to generate rhythmic behavior in DD. While it is possible that light is a necessary cofactor for these self-sustained DN3s to participate in the control of circadian behavior, the hypothesis is favored that it is the DN1s that maintain circadian rhythmicity in LL. This idea is strongly supported by several additional findings. First, the phase of PDP1 molecular oscillations in the DN1s on the third day of LL fits well with the long period of the circadian behavior observed under these conditions in per-overexpressing flies. Second, the behavioral observations made with morgue overexpression also suggest that the critical cells for rhythmicity are not the LNvs, and PER staining in morgue-overexpressing flies gave an independent confirmation that robust circadian molecular oscillations are restricted to the DN1s in LL. Finally, in LNv-rescued cry^b flies, only the DN1s show robust, coherent circadian rhythms in phase with the behavioral rhythms. Remarkably, the DN1s can maintain circadian behavior in LL even when PDF is absent. This indicates that they can work autonomously of LNv output. Interestingly, not all DN1s do oscillate in LL, only about six or seven cells most likely. This shows that the DN1 group is heterogeneous. This is not surprising, since the different groups of circadian neurons were named based on their location in the brain, not on their function or developmental lineage. There is ample evidence for heterogeneity of morphology, gene expression, and behavior within these different groups of cells, including the DN1s (Murad, 2007).

Thus, a subset of DN1s can control and generate circadian behavioral rhythms. They must therefore play an important role in the circadian neuronal circuits. Since ablation of the M cells and E cells results in flies with no morning and evening activity, and no self-sustained rhythms in DD, this could mean that the DN1s are usually functioning downstream of the M and E cells. This is further supported by the fact that in the absence of the neuropeptide PDF—believed to be the critical synchronizing signal secreted by the M cells—the DN1s cannot maintain their circadian rhythms in the long run in DD. The DN1s can thus probably function as a relay connecting the LNvs with the neurosecretory cells of the pars intercerebralis (PI), believed to play an important role in the control of locomotor behavior. A LNvs-DN1-PI pathway has also been suggested based on the anatomical studies of the projections of the small LNvs and the DN1s. The expression of the receptor for PDF in at least a subset of DN1s also supports the existence of a functional connection between them and the LNvs. The implication of this connection is that, in wild-type flies under LL, the LNvs should constantly send a disruptive signal to the DN1s, presumably the nonoscillating secretion of PDF (Murad, 2007).

This leaves the following question: if the LNvs and rhythmic PDF secretion are normally required for the DN1s to be rhythmic, why are the DN1s able to free themselves from the disruptive effects of constant light, while at the same time becoming independent of the LNvs? The results show that an important mechanism is the inhibition of the CRY-dependent light input pathway. Indeed, morgue-overexpressing flies are defective in the CRY-dependent behavioral responses to short light pulses, and cry loss-of-function mutations also result in rhythms driven by the DN1s. In the case of per overexpression, it is presumed that the TIM role is reduced, since one of its major functions is to protect PER from proteasomal degradation. TIM is the target of CRY; thus its reduced importance would result in DN1s that are less sensitive to the CRY input pathway. In addition, overexpression of Shaggy, which inhibits CRY signaling, also results in LL rhythms driven by dorsal neurons. However, under natural environmental conditions, inhibition of the CRY input pathway is probably not required for the DN1s to participate in the control of circadian rhythms. Indeed, even in the polar regions of the globe that experience constant light conditions during the summer, the elevation of the sun varies during the day, and this should result in variations of temperature sufficient to synchronize the DN1 circadian clock (Murad, 2007).

The mechanism by which the DN1s avoid becoming arrhythmic in LL as a result of the molecular arrhythmicity of the LNvs, which should result in constant PDF secretion, is not clear yet. It is possible that the presence of light inhibits PDF signaling and thus promotes the role of the DN1s. Light input could come from the eyes, ocelli, or from the DN1s themselves. Alternatively, the DN1s could induce rhythmic PDF secretion. The fact that PDF is not required for LL behavioral rhythms does not exclude this possibility, particularly since the robustness of the rhythms is improved by the presence of PDF (Murad, 2007).

Interestingly, per and morgue overexpression results in a very similar long period phenotype under LL, which could suggest that these two molecules coincidentally affect the period length of the circadian molecular pacemaker in the same way. In DD, however, per overexpression does affect behavioral period length, while morgue does not. The long period phenotype observed in LL actually probably reflects the fact that the CRY input pathway is not completely blocked in the DN1s of per- or morgue-overexpressing flies. Indeed, under very low light intensity, wild-type flies exhibit a long period phenotype as well. In addition, morgue overexpression does not completely block the CRY-dependent responses to short light pulses. Finally and most importantly, LNv-rescued cry^b flies (in which the CRY input pathway is completely nonfunctional in the DN1s) have 24 hr period rhythms. The LNv-rescued cry^b flies show nevertheless a higher degree of arrhythmicity than normal cry^b flies or than flies overexpressing morgue or per. This might be due to the desynchronization observed within the DN3 group of circadian neurons. Indeed, the DN3s do not appear to be desynchronized in per- or morgue-overexpressing flies (Murad, 2007).

A previous report had already shown that LNv-rescued cry^b flies are partially rhythmic, and this was interpreted as evidence for a functional role of CRY directly in the LNvs. The new results show that expression of CRY in the LNvs is probably not very important for the response to constant light. The DN1s are the important cells for this response. Does this mean that CRY is not a photoreceptor in the LNvs? It is thought that CRY actually does function as a photoreceptor in the LNvs as well. CRY is expressed in these cells, and LNv-rescued cry^b flies show very significantly rescued responses to short light pulses. Preliminary experiments with morgue overexpression limited to the LNvs confirm a predominant role of these cells for light-pulse responses. Thus, the CRY input pathway might mediate response to short light pulses by its action in the LNvs and constant light responses by its action in the DN1s (Murad, 2007).

In summary, the work underscores the importance of the DN1s in the control of circadian behavior and responses to light. Earlier genetic studies have indicated that the DN1s modulate the sensitivity of the circadian network to light:dark cycles of very low light intensity. The current results significantly extend this observation by showing the profound impact the DN1s have on the response to constant light and by demonstrating that these cells not only modulate circadian light responses but can also become the driving force controlling circadian locomotor behavior, and this in the absence of environmental cues and functional LNvs. This confers upon them a unique status among non-PDF circadian neurons. One of the striking results is that genetically identical flies rely either on the LNvs or the DN1s for the control of their circadian rhythms, depending on the presence or absence of light. Indeed, the LNvs determine period length in these experiments with per overexpression in DD, but in LL the DN1s set the pace. That the presence or the absence of light can so remarkably shift the dominance from one cell group to the other strongly suggests that the relative contributions of the LNvs and DN1s to the control of circadian rhythms change during the course of the year, particularly at high latitude. The DN1s, which interestingly generate evening activity, would play a more prominent role in the control of circadian behavior during the long days of the summer, while the LNvs would be more important when photoperiods are shorter (Murad, 2007).

Light activates output from evening neurons and inhibits output from morning neurons in the Drosophila circadian clock

Animal circadian clocks are based on multiple oscillators whose interactions allow the daily control of complex behaviors. The Drosophila brain contains a circadian clock that controls rest-activity rhythms and relies upon different groups of PERIOD (Per)-expressing neurons. Two distinct oscillators have been functionally characterized under light-dark cycles. Lateral neurons (LNs) that express the pigment-dispersing factor (PDF) drive morning activity, whereas PDF-negative LNs are required for the evening activity. In constant darkness, several lines of evidence indicate that the LN morning oscillator (LN-MO) drives the activity rhythms, whereas the LN evening oscillator (LN-EO) does not. Since mutants devoid of functional Cryptochrome (Cry), as opposed to wild-type flies, are rhythmic in constant light, transgenic flies were analyzed expressing Per or Cry in the LN-MO or LN-EO. Under constant light conditions and reduced Cry function, the LN evening oscillator drives robust activity rhythms, whereas the LN morning oscillator does not. Remarkably, light acts by inhibiting the LN-MO behavioral output and activating the LN-EO behavioral output. Finally, this study shows that PDF signaling is not required for robust activity rhythms in constant light as opposed to its requirement in constant darkness, further supporting the minor contribution of the morning cells to the behavior in the presence of light. It is therefore proposed that day-night cycles alternatively activate behavioral outputs of the Drosophila evening and morning lateral neurons (Picot, 2007).

The PDF-expressing LNs and the PDF-negative LNs were previously characterized as morning and evening cells, respectively, in LD conditions. Furthermore, the morning LNs were able to drive robust 24-h rhythms in DD, whereas evening LNs were not. This study shows that in LL, the evening LNs drive robust rhythms when cryptochrome signaling is absent or reduced, whereas the morning cells are not able to do so. Surprisingly, the molecular oscillations of both groups can be uncoupled from behavioral rhythmicity, depending on light conditions. In DD, the two LN groups show autonomous molecular cycling, but there is no behavioral output when the LN-EO is cycling alone. In LL (and reduced Cry signaling), both groups still show autonomous cycling, but there is no behavioral output when the LN-MO is cycling alone. It is therefore concluded that light has opposite effects on the behavioral output of the two LN oscillators, activating it from the evening LNs and inhibiting it from the morning LNs (Picot, 2007).

The opposite effects of light on the behavioral outputs do not appear to be related to entrainment, since Per oscillations in both the PDF-positive and PDF-negative LNs are synchronized to the LD cycles even in the absence of Cry signaling. The inhibiting effect of light on the LN-MO behavioral output is abolished when the visual system is genetically ablated. This suggests that the projections of the visual system photoreceptors convey, not only input information to the PDF cells (light entrainment), but also signals to control their behavioral output (light inhibition). It is tempting to speculate that light exerts both effects through a direct connection of the PDF cells with the visual system. The Hofbauer-Büchner eyelet photoreceptors that project directly to the LN-MO neurons and participate in the entrainment provide a possible pathway (Picot, 2007).

It was recently reported that the overexpression of Per or of the Shaggy (Sgg) kinase in the PDF-negative clock neurons induced rhythmic behavior in LL. The rhythmicity was associated with the cycling of Per subcellular localization in some of the DNs, whereas the PDF-expressing cells were molecularly arrhythmic. These studies therefore concluded that some DN subsets are able to drive behavioral rhythms in LL. Different groups of PDF-negative cells may thus be able to drive behavioral rhythms in constant light, depending on whether and how the molecular clock is manipulated. Such manipulation could also directly affect molecular oscillations, making them less easy to detect. Since Cry does not appear to have a core clock function in the brain, these data are largely based on situations in which the clock mechanism is little if at all altered. The data support a major contribution of the LN-EO to the robust rhythms of cry^b mutants in LL (Picot, 2007).

The strong rhythmicity of the cry^b pdf⁰ double mutants in LL contrasts with their weak rhythmic behavior in DD. Altogether, these results strongly suggest that this robust rhythm is generated by the LN-EO, which would therefore behave as a PDF-independent autonomous oscillator. However, the period of the oscillator is clearly influenced by PDF signaling, and thus by the LN-MO, going from 24–25 h in cry^b to 22–23 h in cry^b pdf⁰ flies. An attractive possibility is that the strong short-period rhythm observed in the cry^b pdf⁰ double mutant in LL has the same neuronal origin as the weak short-period rhythm described for pdf⁰ mutants in DD. The cellular basis of this PDF-independent oscillator in DD remains unclear, although the presence of similar rhythms in flies genetically ablated for the PDF-expressing neurons suggests that it originates from other clock cells (Picot, 2007).

Different results were obtained for the recently described DN-based LL oscillators. When transferred to a pdf⁰ background, all SGG-overexpressing flies were found to be arrhythmic, whereas about 60% of the Per-overexpressing flies displayed long-period rhythms. This suggests that different types of DNs with different sensitivity to PDF may have been analyzed in these two studies. Although some DNs may contribute to the PDF-independent rhythms, the data suggest a strong contribution of PDF-negative LNs to the rhythmic behavior that persists in pdf⁰ mutants. The weakness of the short-period rhythm of pdf⁰ flies in DD may reflect the inhibition of the LN-EO output in the absence of light (Picot, 2007).

These results indicate that whereas the LN-MO autonomously drives rhythmic behavior in constant darkness, the LN-EO plays this role in constant light, if Cry signaling is abolished or reduced. It is thus suggested that in natural LD conditions, Drosophila behavior could be driven by the LN-MO during the night, and by the LN-EO during the day, when cryptochrome is quickly degraded by light. This supports a model of a light-induced switch between the circadian oscillators of the LNs that would allow a better separation of the dawn and dusk activity peaks in day–night conditions. It has been shown that PDF-expressing LNs drive the clock neuronal network in short days, whereas PDF-negative DN subsets take the lead in long days. Thse results suggest that the PDF-negative cells of the LN-EO could also be a major player during the long days. Surprisingly, it was found that light does not seem to act on the molecular oscillations, but inhibits the LN-MO behavioral output and promotes the LN-EO behavioral output, which may provide an efficient fine tuning of the contributions of the two oscillators. It therefore appears that the visual system controls both the input (entrainment) and the behavioral output of the LN oscillators in the Drosophila brain clock. In species such the honeybee or the flour beetle, which appear to lack a light-sensitive Cry protein, this role of the visual system may be particularly important (Picot, 2007).

Pigment dispersing factor-dependent and -independent circadian locomotor behavioral rhythms

Circadian pacemaker circuits consist of ensembles of neurons, each expressing molecular oscillations, but how circuit-wide coordination of multiple oscillators regulates rhythmic physiological and behavioral outputs remains an open question. To investigate the relationship between the pattern of oscillator phase throughout the circadian pacemaker circuit and locomotor activity rhythms in Drosophila, the electrical activity and pigment dispersing factor (PDF) levels of the lateral ventral neurons (LNv) were perturbed, and their combinatorial effect on molecular oscillations was assayed in different parts of the circuit and on locomotor activity behavior. Altered electrical activity of PDF-expressing LNv causes initial behavioral arrhythmicity followed by gradual long-term emergence of two concurrent short- and long-period circadian behavioral activity bouts in ~60% of flies. Initial desynchrony of circuit-wide molecular oscillations is followed by the emergence of a novel pattern of period (PER) synchrony whereby two subgroups of dorsal neurons (DN1 and DN2) exhibit PER oscillation peaks coinciding with two activity bouts, whereas other neuronal subgroups exhibit a single PER peak coinciding with one of the two activity bouts. The emergence of this novel pattern of circuit-wide oscillator synchrony is not accompanied by concurrent change in the electrical activity of the LNv. In PDF-null flies, altered electrical activity of LNv drives a short-period circadian activity bout only, indicating that PDF-independent factors underlie the short-period circadian activity component and that the long-period circadian component is PDF-dependent. Thus, polyrhythmic behavioral patterns in electrically manipulated flies are regulated by circuit-wide coordination of molecular oscillations and electrical activity of LNv via PDF-dependent and -independent factors (Sheeba, 2008).

The period values of the short and long-period activity bouts (~22.5 and ~25 h) observed in these studies closely match those reported earlier for several clock mutants of Drosophila. The fact that robust multiple behavioral rhythms are observed in numerous mutant backgrounds strongly suggests that homeostatic behavioral resynchrony to period values of ~22.5 and ~25 h is a circuit-level constraint rather than a property of any single subgroup of clock neurons. Nevertheless, it is noted that the two periods that emerge in the mutant cry^b under dim LL undergo continuous and opposite changes in period length with increasing light intensity. Thus, it is likely that inputs perceived by individual components of the pacemaker circuit may differentially influence components of the circuit during the intermediary or transition states, whereas the steady state period is determined by the entire network. Although many studies have proposed the dominance of the sLNv in determining the free-running rhythm in DD, previous reports suggest that the molecular oscillations in DN1 neurons play an important role in determining rhythmic activity/rest behavior both under DD and LL conditions. The results of a study which manipulated molecular oscillations in circadian pacemaker circuit by targeted expression of Shaggy (Sgg; a clock component whose overexpression speeds up the oscillations in mRNA of circadian genes) suggests that whereas sLNv, LNd, DN1, and DN3 cells are part of a circuit that regulates locomotor activity rhythm, the lLNv and DN2 cells form a separate and independent circuit that apparently does not influence locomotor activity rhythm and that the DN2 cells are the dominant component of this second circuit and regulate the oscillations in the lLNv (Sheeba, 2008).

Is there a clear association with the phase of molecular oscillation in any of the circadian pacemaker neurons with the phase of behavioral locomotor activity? Under LD, wild-type flies show two peaks in locomotor activity, yet, all known pacemakers exhibit a synchronized phase of Per oscillation that peaks coincident with the morning peak in activity. Wild-type flies when subjected to DD show a single peak in activity (apparently a derivative of the evening peak), and the peak in Per levels of all cells remain synchronized for the first few days, occurring at the trough of behavioral activity. After ~5 d in DD, the phase of peak Per levels appears to drift apart among the members of the circuit, with DN2 cells being out-of-phase, as determined by sampling at 12 h or 6 h intervals. When Per is overexpressed in the pacemaker circuit using the tim-GAL4 driver, it is possible to induce a robust single-period rhythm under LL in >90% of flies. In this case, molecular oscillation appears to persist in DN1 up to the fourth day in LL as quantified by cell counts based on PDP-1 staining. The peak in PDP-1-immunopositive cell numbers coincides with falling levels of locomotor activity (although not the trough). Two simultaneously occurring behavioral rhythms are seen in LL in cry^b mutants, with increasing fraction of flies showing polyrhythmic locomotor behavior with increasing light intensity. Per levels in the PDF positive sLNv and the PDF negative fifth sLNv are antiphasic as determined by sampling at time points (12 h intervals) corresponding to the peak activity of each of the two polyrhythmic bouts in LL in cry^b mutants and a subset of LNd appears to be in phase with the PDF negative fifth sLNv and has high Per levels during one of two activity peaks (the short-period bout apparently derived from the LD morning peak). For flies expressing voltage-gated sodium channel (NaChBac) in the LNv, the phase distribution of Per cycling between the different pacemaker neuronal subgroups shows an antiphasic relationship between the PDF-positive and PDF-negative subsets of sLNv, but is opposite to that seen in cry^b flies under LL (sampled at ~6 h intervals). Furthermore, two peaks in PER levels are seen in DN1 and DN2 and also a clear oscillation in LNd that is not seen in controls or any other study in prolonged DD. Thus, the association between the short-period and long-period activity bouts with the PDF positive and negative sLNv is labile and is highly dependent on environmental or intercellular signals. Furthermore, the polyrhythmicity in behavior and novel pattern of synchrony among the different members of the circadian pacemaker circuit in NaChBac flies appears to be a result of the alteration of electrophysiological properties in either the sLNv, lLNv, or both types of LNv (Sheeba, 2008).

To summarize what has been observed for the temporal relationship between clock cycling in the pacemaker neurons and overt locomotor behavior, from these results and those from previous studies, it is concluded that there is no absolute relationship between activity level and the phase of Per cycling in sLNv. This is particularly clear when comparing the experimental conditions which lead to two behavioral activity peaks, including the morning and evening bouts for wild type flies in LD; and polyrhythmic bouts seen for LL-cry^b; DD-NaChBac expressed in the LNv. Furthermore, it is proposed that the relationship between the pattern of molecular oscillations among the different neuronal subgroups and rhythmic activity rest behavior in the absence of external time cues under DD and LL is likely to be a result of continuous recalibration of signals being perceived by members of the pacemaker circuit. When these signals are below a certain threshold the circuit remains fairly tightly synchronized in terms of the molecular oscillation of clock proteins. Whereas when membrane electrical properties of parts of the circuit are altered to produce higher-than-threshold signals, molecular oscillations in the circuit are rendered asynchronous along with a loss of temporally regulated behavioral activity (Sheeba, 2008).

The circuit gradually recovers from perturbations applied to parts of the network via circuit-wide plasticity as demonstrated by the emergence of novel behavior pattern with novel consolidated activity patterns and synchrony in molecular oscillation in different subgroups of the circuit. The electrically perturbed LNv do not recover their normal pattern of spontaneous action potential firing. Previous work shows that current injection evokes firing in otherwise silent large LNv. The absence of spontaneous firing in this previous study is probably caused by technical differences in the recording procedures. The results of the current study provide empirical evidence for the idea that gradual resynchrony among a large number of constituent oscillators could account for initial arrhythmicity and subsequent emergence of rhythmic behavior seen in animals when exposed to LL. More recent studies in mammals indicate a mechanism for plasticity in neural networks and outputs involving emergence of a coherent oscillation from previously asynchronous oscillator cells in sub areas of SCN when animals are transferred to DD from LL. In summary, the adult circadian pacemaker circuit exhibits electrical-activity dependent circuit-level plasticity of oscillators as shown by the reorganization of molecular oscillation and rhythmic activity/rest behavior. Electrophysiological studies of pacemaker cells in the accessory medulla of cockroaches has led to the hypothesis that pacemaker cells are organized into assemblies with distinct phases which are synchronized by neurotransmitters such as PDF and GABA. In Drosophila this study shows clear evidence for the existence of PDF-independent factors that contribute toward the regulation of circadian activity/rest rhythm also suggesting the existence of compensatory/redundant mechanisms in the pacemaker neuronal circuit. It is hypothesized that the emergence of rhythmic activity due to NaChBac expression using pdfGAL4 driver in pdf⁰¹ flies may cause the rhythmic release of yet unknown neurotransmitters that are otherwise released at lower levels than would be necessary to elicit robust rhythmic behavior. Electrophysiological measurements of NaChBac expressing flies have shown that lLNv show giant action potentials, which may likely trigger downstream neurons to release, signals that can compensate for the lack of PDF. Together, the results of the current experiments along with those of others that have examined the relationship between activity and the pattern of phase distribution among the different pacemaker components suggests that different genetic manipulations and environmental conditions place the architecture of the pacemaker circuit to unique steady states, each of which is able to use different components of the circadian pacemaker circuit to generate distinct circadian patterns in behavior (Sheeba, 2008).

Circadian control of membrane excitability in Drosophila melanogaster lateral ventral clock neurons

Drosophila circadian rhythms are controlled by a neural circuit containing ~150 clock neurons. Although much is known about mechanisms of autonomous cellular oscillation, the connection between cellular oscillation and functional outputs that control physiological and behavioral rhythms is poorly understood. To address this issue, whole-cell patch-clamp recordings were performed on lateral ventral clock neurons (LN_vs), including large (lLN_vs) and small LN_vs (sLN_vs), in situ in adult fly whole-brain explants. Two distinct sizes of action potentials (APs) were found in >50% of lLN_vs that fire APs spontaneously; large APs originate in the ipsilateral optic lobe and small APs in the contralateral. lLN_v resting membrane potential (RMP), spontaneous AP firing rate, and membrane resistance are cyclically regulated as a function of time of day in 12 h light/dark conditions (LD). lLN_v RMP becomes more hyperpolarized as time progresses from dawn to dusk with a concomitant decrease in spontaneous AP firing rate and membrane resistance. From dusk to dawn, lLN_v RMP becomes more depolarized, with spontaneous AP firing rate and membrane resistance remaining stable. In contrast, circadian defective per⁰ null mutant lLN_v membrane excitability is nearly constant in LD. Over 24 h in constant darkness (DD), wild-type lLN_v membrane excitability is not cyclically regulated, although RMP gradually becomes slightly more depolarized. sLN_v RMP is most depolarized around lights-on, with substantial variability centered around lights-off in LD. These results indicate that LN_v membrane excitability encodes time of day via a circadian clock-dependent mechanism, and likely plays a critical role in regulating Drosophila circadian behavior (Cao, 2008).

This study investigated the temporal regulation of membrane excitability of PDF-expressing LN_v clock neurons. RMP and AP firing patterns in lLN_vs were characterized, and it was demonstrated that lLN_v membrane excitability oscillates as a function of time of day in LD. This oscillation requires a functional circadian oscillator, as it is abolished in per⁰ flies. This requirement could be cell autonomous to the lLN_vs, as suggested by the fact that the oscillation is abolished in DD, a condition in which lLN_v, but not other clock neuron, transcriptional oscillation ceases. sLN_v RMP is most depolarized near lights-on (Cao, 2008).

lLN_v spontaneous activity is somewhat variable, with 33% of cells silent in the light phase and 38% in the dark in LD. That, overall, 35% of lLN_vs are silent in LD is slightly higher but still comparable with mammalian clock neurons in the suprachiasmatic nucleus (SCN). Despite the 35% of silent lLN_vs, the average AP firing frequency and the percentage of lLN_vs that fire APs show a circadian temporal pattern similar to that in mammalian SCN, which is consistent with linear regression analysis of the scatter plot of individual cells. Interestingly, in contrast to the cyclic changes in lLN_v RMP, membrane resistance and AP firing rate decrease from dawn to dusk in the day but stay low during the night in LD. The biophysical basis for this uncoupling of RMP from AP firing rate and membrane resistance at night is not clear, but presumably reflects differential modulation of particular ion channel subtypes. This issue bears further investigation in the future using physiological, pharmacological, and genetic techniques (Cao, 2008).

lLN_vs exhibit two sizes of APs, with the small APs approximately half the amplitude of the large APs. Gap junction inhibitors reduce the frequency of small APs and alter the normal firing rate of large APs, suggesting that gap junctions are involved in modulating membrane excitability of lLN_vs. This is consistent with observations of cockroach lateral neurons, where gap junction blockers also influence spontaneous AP firing rate, and clock neurons in mammalian SCN. However, none of the three gap junction blockers had any effect on the amplitude of either large or small APs, thus making it highly unlikely that APs generated in coupled cells are propagating passively to the site of recording at the soma through gap junctions. That cutting the posterior optic tract removes small APs strongly supports that small APs originate in the contralateral optic lobes. This raises the interesting possibility that the ipsilateral and contralateral arbors of individual lLN_vs can process information independently (Cao, 2008).

WT lLN_v membrane excitability is modulated as a function of time of day. In particular, in the light phase of LD, lLN_v RMP becomes more hyperpolarized from dawn to dusk, as the AP firing rate and lLN_v membrane resistance decrease. In the dark phase, lLN_v RMP becomes more depolarized from the beginning to the end of the night, whereas the AP firing rate and membrane resistance stay relatively stable. This continuous cyclical modulation of WT lLN_v RMP is consistent with previous coarse time-scale studies demonstrating that lLN_v RMP is more depolarized in the early morning and more hyperpolarized in the early night (Park, 2006; Sheeba, 2008). This temporal modulation is almost completely abolished in per⁰ flies, thus demonstrating for the first time its dependence on an intact transcriptional feedback oscillator. Although this is consistent with an autonomous requirement for transcriptional feedback oscillation in the lLN_vs themselves, because per⁰ flies lack transcriptional feedback oscillation in all neurons, this could be a circuit-based phenomenon. To further address this question, the modulation of WT lLN_v excitability was examined on the first day in DD conditions (DD-D1), in which transcriptional oscillation in lLN_vs, but not in any other clock neurons, is abolished. The absence of oscillatory modulation of lLN_v excitability in DD-D1 further supports the conclusion that transcription feedback oscillation is required cell-autonomously in the lLN_vs, and the transcriptional feedback oscillator directly modulates lLN_v membrane excitability at the cellular level. The apparent difference between the demonstration of an absence of dependence of electrical excitability on time of day on DD-D1, and Sheeba's (2008) demonstration of presence of a dependence of electrical excitability on time of day on DD-D14 actually makes sense in light of what is known about the response of lLN_vs to constant darkness. Whereas sLN_vs and other clock neurons maintain their cellular rhythmicity immediately after transition from LD to DD conditions, the lLN_vs immediately lose their cellular rhythms with transition into DD, and only regain cellular rhythmicity after several days in DD. This immediate loss of lLN_v cellular rhythmicity followed by resumption several days later perfectly accounts for the fact that no rhythm of lLN_v electrical excitability was observed on DD-D1 whereas Sheeba (2008) observed such a rhythm on DD-D14 (Cao, 2008).

Either pdf gene mutation, LN_v ablation, or LN_v electrical silencing severely disrupts free-running locomotor rhythms, indicating that PDF-expressing LN_vs play a critical role in controlling circadian behavior. Both sLN_vs and lLN_vs express PDF. However, previous studies suggest that of these two cell groups it is the sLN_vs that are most important for controlling free-running rhythms in DD. In particular, restoration of per gene expression in sLN_vs, but not lLN_vs can rescue free-running circadian rhythmicity in per⁰ null mutant flies. Furthermore, as mentioned above, lLN_v transcriptional feedback oscillation cease in DD. The lLN_vs have extensive dendritic arborizations in the medulla and project to contralateral lLN_vs through the posterior optic tracts, thus enabling enable them to potentially receive light information and synchronize the two hemispheres. The lLN_vs also project to the accessory medulla (aMe), where the sLN_vs have short dendrites that may be postsynaptic to lLN_v projections. Although the functional role of lLN_vs in circadian rhythms is mostly unknown, it is reasonable to speculate based on this anatomy that lLN_vs relay environmental light information to the sLN_vs via synapses formed in aMe. It is thus proposed that circadian modulation of lLN_v membrane excitability in LD gates the transfer of entraining light information to the sLN_vs, which more directly control circadian locomotor rhythms (Cao, 2008).

Because sLN_vs are thought to be pacemakers of the neural circuit controlling circadian rhythms, it is critical to understand how membrane excitability of sLN_vs is regulated, and how sLN_v membrane excitability is transformed to signals that control downstream neurons, and, ultimately, circadian locomotor activity. Recording was performed of 103 sLN_vs in LD, and it was found that sLN_v RMP is most depolarized around lights-on, with greater variability around lights-off. The two linear regression fits were selected to span the 6 h just before, and just after, lights-on (ZT0, ZT24). sLN_v RMP exhibits significant trends of depolarization before lights-on and hyperpolarization after lights-on. It is clear that during the 12 h surrounding lights-off (ZT6-ZT18), there is much more variability in sLN_v RMP. This difference makes some sense, as sLN_vs are considered to be the 'morning' cells that drive the increase in locomotor activity that anticipates lights-on. This would be consistent with an increase in sLN_v electrical excitability in the hours preceding lights-on, and a decrease after lights-on, as was clearly observed. Because the sLN_vs are not thought to be important for driving the increase in locomotor activity that anticipates lights-off (ZT12), it is perhaps not surprising that there are no consistent trends in sLN_V electrical excitability surrounding lights-off (ZT12). The reason sLN_vs exhibit little spontaneous activity in this brain explant preparation is not clear, but it may reflect technical issues relating to the extremely small size of their soma. Previous studies indicate that the peak of anti-PDF staining in the sLN_v nerve terminals is around lights-on, and the trough is around lights-off. It is thus possible that the trough of PDF accumulation around lights-off represents depletion of the releasable pool of PDF starting in the early day, when sLN_vs are most depolarized (Cao, 2008).

This study, and that by Sheeba (2008), demonstrate a dependence of lLN_V electrical excitability on time of day in LD and after many days in DD, respectively. The results go beyond those of Sheeba (2008) in a number of important ways, including demonstration that the daily rhythm of lLN_V electrical excitability depends on a functional cellular circadian oscillator, demonstration of a daily rhythm of electrical excitability in the sLN_V pacemaker subset of clock neurons, and elucidation of the cellular origin of the bimodal distribution of sizes of lLN_V APs. Future studies will determine the daily rhythms of electrical excitability of other functionally and anatomically identifiable subsets of fly clock neurons (Cao, 2008).

The GABA_A receptor RDL acts in peptidergic PDF neurons to promote sleep in Drosophila

Sleep is regulated by a circadian clock that times sleep and wake to specific times of day and a homeostat that drives sleep as a function of prior wakefulness. Flies display the core behavioral features of sleep, including relative immobility, elevated arousal thresholds, and homeostatic regulation. Sleep-wake modulation was assessed by a core set of circadian pacemaker neurons that express the neuropeptide PDF. It was found that disruption of PDF function increases sleep during the late night in light:dark and the first subjective day of constant darkness. Flies deploy genetic and neurotransmitter pathways to regulate sleep that are similar to those of their mammalian counterparts, including GABA. RNA interference-mediated knockdown of the GABA_A receptor gene, Resistant to dieldrin (Rdl), in PDF neurons reduces sleep, consistent with a role for GABA in inhibiting PDF neuron function. Patch-clamp electrophysiology reveals GABA-activated picrotoxin-sensitive chloride currents on PDF+ neurons. In addition, RDL is detectable most strongly on the large subset of PDF+ pacemaker neurons. These results suggest that GABAergic inhibition of arousal-promoting PDF neurons is an important mode of sleep-wake regulation in vivo (Chung, 2009).

It is proposed that GABA release inhibits large LNv output and PDF release to reduce wake, suggesting an important role for GABA inhibition. In this model, the circadian clock times PDF neuron activation and PDF release during the late night and following day to promote waking behavior. Of note, a similar arousal-promoting function for circadian pacemaker neurons has been described in mammals. This is also approximately the time when the large LNv have been shown to be more depolarized and have higher levels of spontaneous activity. RDL receptors on LNv soma and on fibers in the accessory medulla suggest that GABA may regulate LNv excitability. It is interesting that GABA is also an important neurotransmitter in mammalian circadian pacemaker neurons, capable of reducing their spontaneous activity. In addition, RDL receptors on PDF varicosities in the optic lobe may function presynaptically to regulate PDF release. GABA may also act through metabotropic GABA_B receptors, which have been described in the sLNv, but their function in circadian or sleep behavior is unknown. GABAergic signaling may affect the function of the transcription factor ATF2, which is important for PDF neuron function in sleep. Changes in PDF neuron function may in turn act by antagonizing sleep-promoting circuits that exist within the mushroom bodies as well as the pars intercerebralis (PI). Of note, the PI appears to express the PDF receptor. Identifying the anatomic targets of PDF as well as the neural sources of GABAergic inputs will be important for further defining sleep-wake circuits in Drosophila (Chung, 2009).

Comparative analysis of Pdf-mediated circadian behaviors between Drosophila melanogaster and D. virilis

A group of small ventrolateral neurons (s-LN(v)'s) are the principal pacemaker for circadian locomotor rhythmicity of Drosophila melanogaster, and the pigment-dispersing factor (Pdf) neuropeptide plays an essential role as a clock messenger within these neurons. In comparative studies on Pdf-associated circadian rhythms, it was found that daily locomotor activity patterns of D. virilis were significantly different from those of D. melanogaster. Activities of D. virilis adults are mainly restricted to the photophase under light:dark cycles and subsequently became arrhythmic or weakly rhythmic in constant conditions. Such activity patterns resemble those of Pdf⁰¹ mutant of D. melanogaster. Intriguingly, endogenous D. virilis Pdf (DvPdf) expression was not detected in the s-LN(v)-like neurons in the adult brains, implying that the Pdf(01)-like behavioral phenotypes of D. virilis are attributed in part to the lack of DvPdf in the s-LN(v)-like neurons. Heterologous transgenic analysis showed that cis-regulatory elements of the DvPdf transgene are capable of directing their expression in all endogenous Pdf neurons including s-LN(v)'s, as well as in non-Pdf clock neurons (LN(d)'s and fifth s-LN(v)) in a D. melanogaster host. Together these findings suggest a significant difference in the regulatory mechanisms of Pdf transcription between the two species and such a difference is causally associated with species-specific establishment of daily locomotor activity patterns (Bahn, 2009).

This study has characterized circadian locomotor activity rhythms of D. virilis, a species that has radiated from the melanogaster linage ~63 million years ago, approximately the beginning of the Cenozoic era. During this geological period, major paleoclimatic changes are proposed to drive speciation of Drosophila. In addition, studies indicate diversification of native habitats of Drosophila species, as D. virilis is suggested to be indigenous to eastern Asia, whereas D. melanogaster is believed to be African origin. Therefore, it is possible that D. virilis and D. melanogaster may have evolved unique biological clock systems that suit their endemic environment. This is supported by behavioral data that revealed substantially different daily and circadian locomotor activity patterns between D. virilis and D. melanogaster (Bahn, 2009).

The first insight that Pdf might be responsible for behavioral characteristics of D. virilis comes from the uncanny resemblance in locomotor activity patterns between D. virilis and Pdf⁰¹ mutant flies of D. melanogaster. Under LD condition, activities of the Pdf⁰¹ flies are largely restricted to the daytime; such diurnally shifted activity of the Pdf⁰¹ flies is likely to be attributed to both prominently reduced morning anticipatory behavior and the slight phase advance of evening activity peaks to the photophase. Moreover, like D. virilis, Pdf⁰¹ flies are largely arrhythmic or weakly rhythmic in DD condition. Therefore, it is reasonable to suggest that the lack of Pdf expression in the s-LN_v equivalent neurons is intimately associated with behavioral characteristics of D. virilis. These results are also consistent with morning oscillator functions of s-LN_v's, and further support the importance of Pdf's role within the s-LN_v neurons for lights-on anticipatory behavior (Bahn, 2009).

Daily locomotor activity patterns described for the housefly, M. domestica are notably similar to those of D. virilis, as both species display day-phase-restricted activity without lights-on anticipation. IHC using anti-Pdf showed both large and small LN_v-equivalent neuronal groups in the adult brain of M. domestica. In contrast to this result, in situ hybridization revealed MdPdf mRNA expression only in the l-LN_v-like neurons. These results were confirmed independently. A plausible explanation for this discrepancy is that the s-LN_v-like neurons contain materials that cross-react with the anti-Pdf. From these data, it is tempting to propose that lack of Pdf expression in s-LN_v-like neurons is also responsible for diurnally active locomotion displayed by M. domestica (Bahn, 2009).

In the heterologous host, expression of the DvPdf gene is evident in all of DmPdf-positive groups, suggesting that donor DvPdf regulatory elements are capable of interacting with host trans-acting factors to activate its expression in a manner similar to that of DmPdf. Although no useful information about potential elements emerged from simple sequence alignment between 0.5-kb upstream sequence of DmPdf and 1.9-kb of DvPdf, the cis-regulatory element(s) responsible for s-LN_v Pdf expression is likely conserved in both DmPdf and DvPdf. An important question raised from these studies is, then, Why do D. virilis flies lack DvPdf expression particularly in the s-LN_v-like neurons? It could be that D. virilis does not possess the s-LN_v-like neurons. However, this is unlikely, because a fly species (M. domestica), which is even more remotely related to D. melanogaster, contains Pdf-ir s-LN_v-like neurons, although such immunoreactivity likely originated from cross-reactivity, as mentioned earlier. Attempts were made to confirm the presence of s-LN_v's in the D. virilis CNS using anti-Tim, as was done for D. melanogaster. However, no immunosignals were detectable even in the l-LN_v's at two different time points. Although similarity of the Tim between the two species is substantial (76% overall amino acid identity, perhaps diversity between the two proteins does not allow anti-Tim to detect virilis Tim protein (Bahn, 2009).

In D. melanogaster, DmClk and DmCyc proteins are well-defined upstream positive factors responsible for DmPdf expression specifically in the s-LN_v's. This study study shows that these factors are also essential for the DvPdf transgenic expression in the s-LN_v's, LN_d's and fifth s-LN_v, suggesting that DvClk and DvCyc likely act as positive regulators for the DvPdf in the s-LN_v-like neurons in D. virilis brain. Thus the lack of DvPdf expression in the s-LN_v-like neurons might be due to a loss of function of these proteins in D. virilis (Bahn, 2009).

According to the genome database, DvClk gene (Dvir\GJ11427) predicts to encode a protein of 988 amino acids. RT-PCR result suggests that DvClk encodes a 987-amino-acid product and has differences from the Dvir\GJ11427 at three sites. Two of them are within polyglutamate (Q) stretches, missing two Q's at one position and having the addition of one Q at another position. The other one is a homologous substitution of leucine to isoleucine (data not shown). Amino acid composition of the DvClk shows 70% identity to DmClk. For Cyc, sequence of DvCyc deduced from RT-PCR matches perfectly to that from genome database (Dvir\GJ14003), and amino acid residues of the DvCyc share 85% identity with the DmCyc. In other words, no significant mutations were found within the ORFs of both DvClk and DvCyc that might alter their functions. Moreover, robust activity rhythms displayed by D. virilis under LD cycles, in contrast to significantly abnormal LD behavior of D. melanogaster Clk^Jrk and cyc⁰ mutants, suggest that functions of the two clock proteins are unlikely defective in D. virilis (Bahn, 2009).

Absence of s-LN_v-specific DvPdf expression could be accomplished through negative regulation. According to published work, l-LN_v's and LN_d's in D. melanogaster appear to be originated from the common precursor cells; clusters of these cells are mixed without clear anatomical distinction in the ventral region of early pupal brain. As the pupal development progresses, presumptive LN_d's are separated from l-LN_v's, migrate dorsally, and start to develop their characteristic projections. Shortly after this stage, l-LN_v's become Pdf-positive, while LN_d's remain Pdf-negative. However, one exceptional specimen was found in which the migration of the LN_d's is impaired; interestingly, these neurons are Pdf-positive. These findings are interpreted as follows: activation of the DmPdf in both l-LN_v's and LN_d's during pupal development is a default pathway, and then the suppression of the DmPdf is acquired during the maturation of the LN_d neurons, perhaps through the activation of repressors. These studies provide an interesting possibility of the transcriptional suppression of DmPdf in the LN_d's and fifth s-LN_v. This notion is supported by the ectopic DvPdf transgene expression in these neurons, as negative trans-acting factors might be unable to interact with DvPdf's regulatory region due to sequence incompatibility, thus allowing ectopic expression of the DvPdf. As an extrapolation of these results, it would be interesting to investigate whether negative factors suppress DvPdf expression in the s-LN_v-like (and perhaps LN_d- and fifth s-LN_v-like) neurons in D. virilis. Transgenic dissection of the 1.9-kb DvPdf upstream region will help reveal specific cis-acting elements that are necessary for such negative DvPdf regulation (Bahn, 2009).

A role for blind DN2 clock neurons in temperature entrainment of the Drosophila larval brain

Circadian clocks synchronize to the solar day by sensing the diurnal changes in light and temperature. In adult Drosophila, the brain clock that controls rest-activity rhythms relies on neurons showing Period oscillations. Nine of these neurons are present in each larval brain hemisphere. They can receive light inputs through Cryptochrome (CRY) and the visual system, but temperature input pathways are unknown. This study investigated how the larval clock network responds to light and temperature. Focus was placed on the CRY-negative dorsal neurons (DN2s), in which light-dark (LD) cycles set molecular oscillations almost in antiphase to all other clock neurons. The phasing of the DN2s in LD depends on the pigment-dispersing factor (PDF) neuropeptide in four lateral neurons (LNs), and on the PDF receptor in the DN2s. In the absence of PDF signaling, these cells appear blind, but still synchronize to temperature cycles. Period oscillations in the DN2s were stronger in thermocycles than in LD, but with a very similar phase. Conversely, the oscillations of LNs were weaker in thermocycles than in LD, and were phase-shifted in synchrony with the DN2s, whereas the phase of the three other clock neurons was advanced by a few hours. In the absence of any other functional clock neurons, the PDF-positive LNs were entrained by LD cycles but not by temperature cycles. These results show that the larval clock neurons respond very differently to light and temperature, and strongly suggest that the CRY-negative DN2s play a prominent role in the temperature entrainment of the network (Picot, 2009).

Although the absence of PDF severely affects Drosophila activity rhythms in DD, the exact function of the neuropeptide in the adult clock neuronal network remains unclear. In LD, PDF is required to produce a morning activity peak and to properly phase the evening peak, but not to entrain the brain clock. The behavioral phenotypes of PDF receptor mutants resemble that of the pdf⁰¹ mutant. PDFR is expressed in all clock neurons except the large ventral lateral neurons (l-LNvs), supporting a role of PDF in maintaining phase coherence within the adult clock network in DD. The loss of PER oscillations in the DN2s of pdf⁰¹ larvae demonstrates a clear and novel role of PDF in transmitting not only phase information but also a synchronizing signal without which the receiving neurons are not entrained in LD (Picot, 2009).

The current results show that the CRY-less DN2s are 'blind' neurons that perceive light indirectly. The PDF receptor rescue experiments strongly suggest that PDF acts on its receptor on the larval DN2s themselves, which are located in the vicinity of the LN axon terminals. Furthermore, DN2s possess a wide and dense neuritic network that borders on the axons of the LNs over a large fraction of their length. However, it cannot be ruled out that expression of the receptor in the (PDF-negative) fifth LN is involved in synchronizing the DN2s downstream, through PDF-independent mechanisms (Picot, 2009).

The PDF-negative fifth LN is also a CRY-negative clock neuron, but it cycles in phase with the CRY-positive neurons of the larval brain. The visual input to the PDF-expressing LNs appears sufficient to phase them normally even in cry^b mutants. It could thus be expected to entrain the CRY-less fifth LN in phase with the other larval LNs, as observed, in contrast to the CRY-less DN2s. A direct input from the visual system to the fifth LN is also consistent with its PDF-independent entrainment by LD cycles. Similarly, light entrainment of the larval DN1s in cry^b mutants is consistent with their suggested connection to the visual system. Thus, the CRY-less DN2s would be the only larval clock neurons devoid of such a connection (Picot, 2009).

Adult eclosion rhythms depend on the PDF-expressing LNs and appear to require the PDF-dependent clock that resides in the prothoracic gland. Since the larval DN2s project in the pars intercerebralis, a region of the brain that sends projections to the prothoracic gland, they could play a role in this physiologically important clock function. These results raise the possibility that the damped PER oscillations in the DN2s of the pdf⁰¹ mutants participate to their eclosion phenotype (Picot, 2009).

The DN2s are the only larval clock neurons that are phased identically by light and temperature, but their temperature entrainment appears independent of any LN-derived signal. PER oscillations in the DN2s have a larger amplitude in HC cycles than in LD cycles, also suggesting a prominent role of temperature in their entrainment. Conversely, the molecular oscillations of the PDF-positive LNs have a larger amplitude in LD compared with HC cycles. In the latter, the molecular oscillations of the PDF-expressing LNs seem to follow those in the DN2s, with a large phase change compared with LD conditions. The DN1s and the PDF-negative fifth LN, in contrast, share another phase that is slightly advanced. Interestingly, behavioral and transcriptome data in adult flies indicate that HC cycles result in a general phase advance relative to LD cycles. Cooperative synchronization of the clock by light and temperature likely requires temperature changes to act earlier than light changes since changes in temperature always lag behind changes in solar illumination in nature. The very different relative phasing of the larval clock neurons in HC versus LD cycles suggests different ecological constraints on this life stage, spent mostly burrowed in food, in which light may be a weaker Zeitgeber, and in which the lag between temperature and light changes may be quite different (Picot, 2009).

When a functional clock is absent from the DN2s (and the fifth LN), the larval PDF-expressing LNs are unable to entrain to thermocycles, whereas they autonomously entrain to LD cycles. It remains possible that autonomous temperature entrainment of the larval LNs (but not the DN2s) requires per transcriptional regulation, which the GAL4-UAS system is lacking. But the results demonstrate the existence of a control exerted on the LN clock by CRY-negative clock cells when temperature is the synchronizing cue. Although a role of the fifth LN cannot be ruled out, the absence of autonomous photoperception by the DN2s nicely fits with a role in temperature entrainment. The high cycling amplitude of the DN2s in thermocycles and the locking of the phase of the LNs on that of the DN2s in these conditions strongly support their role in the temperature entrainment of the LNs (Picot, 2009).

Additional studies should investigate whether the DN2s communicate with the LNs via fibers that appear to run along the dorsal projection of the LNs. Alternatively, the dense dendritic-like network of the DN2s could ensure reciprocal exchanges between them and the LNs. A model is thus proposed whereby, in the larval brain, the DN2s and the four PDF-positive LNs form a distinct subnetwork, with the LNs entraining the DN2s in LD, whereas the opposite is true in HC). What becomes of their hierarchy in constant conditions, after entrainment stops? Their relative phases appear to change little at least during the first 2 d after entrainment, whether they have been set in antiphase by LD entrainment, or in phase by HC entrainment. This suggests that, whatever the entraining regimen, the LNs and the DN2s run autonomously in constant conditions. However, it cannot be excluded that one of the two groups still dominates but requires more time after the end of entrainment to shift the phase of the other (Picot, 2009).

The rhythmic behavior of the adult flies that emerge from the temperature-entrained larvae is almost in antiphase compared with the one of flies entrained by light during the larval stage. This strongly suggests that the phase of the adult rhythms is set by the antiphasic oscillations of the larval PDF-positive LNs, consistent with these cells being the only neurons in which molecular cycling persists throughout metamorphosis. It is thus believed that the large phase shift of adult activity can be accounted for simply by the large phase shift of molecular oscillations in the PDF-expressing LNs (Picot, 2009).

It is often assumed that temperature affects the molecular clock directly and identically in all clock cells, as opposed to light, which requires dedicated input pathways. However, in the adult, thermocycles phase the brain clock differently from all peripheral clocks, as judged from whole-tissue oscillations of a luciferase reporter enzyme (Glaser, 2005). Recent data suggest that subsets of clock neurons in the Drosophila adult brain may indeed be dedicated to temperature entrainment. In experiments combining LD and HC entrainment, all DN groups, as well as the less studied lateral posterior neurons (LPNs), seem to preferentially follow thermocycles, whereas the other LNs preferentially follow LD cycles (Miyasako, 2007). Although adult PDF⁺ LNs are able to entrain to thermocycles in the absence of any other functional clock, they do not seem to be required for (and actually slowed down) the temperature entrainment of activity rhythms, whereas the PDF-negative LPNs appear to play a prominent role in such conditions (Picot, 2009).

The current results indicate that a similar specialization toward light or temperature entrainment exists in the larval brain. The DN2s, which appear to be the most temperature-responsive clock neurons, are by themselves completely blind. Conversely, the four PDF-positive LNs, which may be the most light-sensitive clock neurons (with both CRY and the visual system as inputs), appear almost temperature blind, and depend on the DN2s for temperature entrainment. PER-negative DN2s do not allow PER oscillations in the larval LNs, suggesting that entrainment of the latter in HC cycles depends on clock function in the former. The hierarchy of clock neurons thus appears very different during entrainment of the clock network by one or the other Zeitgeber (Picot, 2009).

Identifying specific light inputs for each subgroup of brain clock neurons in Drosophila larvae

In Drosophila, opsin visual photopigments as well as blue-light-sensitive cryptochrome (Cry) contribute to the synchronization of circadian clocks. This study focused on the relatively simple larval brain, with nine clock neurons per hemisphere: five lateral neurons (LNs), four of which express the pigment-dispersing factor (PDF) neuropeptide, and two pairs of dorsal neurons (DN1s and DN2s). Cry is present only in the PDF-expressing LNs and the DN1s. The larval visual organ expresses only two rhodopsins (RH5 and RH6) and projects onto the LNs. PDF signaling is required for light to synchronize the Cry^- larval DN2s. This study shows that, in the absence of functional Cry, synchronization of the DN1s also requires PDF, suggesting that these neurons have no direct connection with the visual system. In contrast, the fifth (PDF^-) LN does not require the PDF-expressing cells to receive visual system inputs. All clock neurons are light-entrained by light-dark cycles in the rh5²;cry^b, rh6¹ cry^b, and rh5²;rh6¹ double mutants, whereas the triple mutant is circadianly blind. Thus, any one of the three photosensitive molecules is sufficient, and there is no other light input for the larval clock. Finally, it was shown that constant activation of the visual system can suppress molecular oscillations in the four PDF-expressing LNs, whereas, in the adult, this effect of constant light requires Cry. A surprising diversity and specificity of light input combinations thus exists even for this simple clock network (Klarsfeld, 2011).

The larval brain clock and its light inputs are generally considered much simpler than their adult counterparts. We find here that larvae, with only nine clock neurons and 12 photoreceptors on each side, nevertheless display four distinct combinations of light inputs (Klarsfeld, 2011).

Anatomical data and the present work show that PDF⁺ LNs are the only brain cells to perceive light both cell autonomously (via CRY) and through a direct connection to the visual system. They thus appear to be the main players responsible for synchronizing the larval brain clock network to LD cycles. The DN2s, in contrast, possess neither type of light input, but play a major role in the temperature entrainment of the clock (Picot, 2009). Previous studies have shown that the DN2s are intrinsically blind and must rely on PDF signaling from the LNs to synchronize to LD cycles (Picot, 2009). This study shows that the other dorsal group, the DN1s, is also sensitive to PDF signaling. In the absence of functional Cry, Pdf is required to synchronize DN1s by light, as demonstrated by the lack of Per oscillations in the DN1s of the cry^b pdf⁰ double mutant. This is consistent with the presence of a dendritic-like arborization from the DN1s close to the dorsal projection of the LNs. On the other hand, it tends to exclude a functional connection between the DN1s and the larval visual system, in agreement with the absence of DN1 neurites reaching the Bolwig's nerve terminals (Klarsfeld, 2011).

The Pdf-dependent entrainment of both DN1s and DN2s by the visual system also indicates that the fifth LN, although projecting largely like the Pdf⁺ LNs, cannot synchronize the DNs. However, the fifth LN might be involved in RH5-dependent acute larval responses to light, which do not require the Pdf⁺ LNs. The entrainment of the fifth LN in the absence of both Cry and the Pdf⁺ LNs suggests a direct connection to the visual system, in agreement with its arborization in the larval optic neuropil. Recent single-cell analysis indeed revealed this arborization to be even broader than that of the Pdf⁺ LNs. However, such connection to the visual system does not allow constant light to disrupt Per oscillations in the fifth LN, contrary to the Pdf⁺ LNs, suggesting different downstream signaling in these two types of visual system targets. Finally, the results suggest a hitherto unsuspected connection between some Cry⁺ neurons and the fifth LN. This connection does not rely on Pdf and could be directly from the DN1s or the Pdf⁺ LNs, which both have projections in the vicinity of the fifth LN's projections (Klarsfeld, 2011).

More generally, the fact that the Cry^- fifth LN and DN2s display normal Per oscillations in the absence of a functional visual system is consistent with Cry transmitting light information in a non-cell-autonomous way. This has already been proposed in the adult brain for the three Cry^- dorsal lateral neurons and the DN2s. However, it remains possible that such nominally Cry^- cells in the adult express very low levels of Cry, as judged from reporter gene expression. In contrast, Cry expression in the larval 5th LN and DN2s was observed neither with antibodies, not with any reporter lines. The present results make it even less likely, because constant light does not affect these neurons at all (Klarsfeld, 2011).

The role of the Pdf neuropeptide in the light entrainment of the DN1s and DN2s appears somewhat different for the two subgroups. First, Pdf sets the DN1s and DN2s to very different phases: the DN2s are set in antiphase with the LNs, whereas the DN1s are set in phase with the LNs. This suggests that the corresponding signaling cascades differ somewhere downstream from the Pdf receptor. In addition, the dispersion of cell labeling intensities suggests that unentrained DN2s oscillate, although asynchronously (even within a single brain hemisphere), while unentrained DN1s do not, but rather express constant, moderate Per and TIM levels. The same may hold true for completely blind larvae. While the LNs and DN2s seem to oscillate with random individual phases, all DN1s display very similar Per levels. This implies that, in LD, Pdf may be needed not only to synchronize but to trigger (or at least maintain until the third larval stage) DN1 oscillations in the absence of Cry activation. In contrast, Pdf synchronizes persistent autonomous oscillations in the DN2s. The non-autonomous cycling of the Cry-expressing DN1s suggests that they may have an important role in synchronizing the network to LD cycles. Conversely, the capacity of the DN2s for autonomous cycling in the absence of light cues may relate to their specific role in temperature entrainment (Klarsfeld, 2011).

Lack of entrainment by light was previously reported for the LNs and the DN1s in norpA^P41;;cry^b larvae, while, rather surprisingly, molecular oscillations were still detected in their DN2s. While the DN2s require Pdf to entrain in LD, they appear to entrain to temperature cycles very efficiently on their own (Picot, 2009). This means one cannot exclude the possibility that, in a previous study, small temperature changes induced by the LD cycles weakly entrained these neurons, but not the others. Alternatively, the DN2s might collect light information from a NORPA-independent pathway. NORPA-independent photoreception appears to participate in adult circadian photoreception (Klarsfeld, 2011).

The results show that RH5, RH6, and Cry are the only light input pathways for synchronizing the larval clock neurons to LD cycles. RH5, RH6, and Cry are each sufficient alone to entrain all these neurons, whereas, in the adult, some clock neurons fail to entrain in the absence of Cry. At least two more rhodopsins, including RH1 and a UV-blue one (RH3 and/or RH4), participate in the adult, so that all available rhodopsins in the adult eye may also be involved in entraining the clock. Recently, at least two classes of larval sensory neurons, outside BO, have been shown to express visual transduction components. One of these two is involved in thermal preferences, with RH1 as the presumed temperature sensor, while the other mediates rhodopsin-independent avoidance of very high light intensities. At least in the conditions used in this study, these novel sensory pathways do not seem to contribute to circadian light entrainment (Klarsfeld, 2011).

Interestingly, constant light, acting Cry independently through the visual system, can abolish or greatly disturb oscillations in the Pdf⁺ LNs of larvae but not adults. Similarly, the larval visual system is required for fast TIM degradation in the LNs at the end of the night. The Pdf⁺ LNs of eyeless adult flies, in contrast, seem to respond normally even to a very short light pulse, suggesting that the visual system is dispensable for the response to light pulses in adults, but not larvae. The different sensitivity of the larval clock to visual system inputs could be related to the change in signaling pathways that occurs as the larval cholinergic visual system develops into the adult histaminergic visual system. Moreover, contrary to the adult situation, the larval visual nerve may be light sensitive all along its length, down to its connection with the LNs, as judged from RH6 and NORPA expression. How visual system signaling ultimately affects the clock, whether in larvae or adults, remains to be discovered (Klarsfeld, 2011).

Both RH5⁺ and RH6⁺ BO photoreceptors contribute to the light responses of the larval brain clock that were tested, i.e., entrainment in LD and disruption of LN rythmicity in LL. Similarly, both photoreceptor types are equally able to suppress TIM levels in the LNs after a 2 h light exposure at the beginning of the night. In contrast, RH5 fibers alone specifically mediate acute larval responses to light, while RH6 fibers alone are specifically required for the development of a serotonergic arborization that also contacts the LNs. That RH6 activation strongly disrupts molecular oscillations in the LNs even in RR was, however, not anticipated. In the adult, RR does not affect molecular or activity rhythms (Klarsfeld, 2011).

Activation of RH6 above 600 nm is less than a few percent of peak activation at ~510 nm. This suggests that the clock of the larval LN is extremely sensitive to red light, which may explain why no larval activity rhythm was recorded in a study that used video tracking in constant red light. A strong sensitivity of larvae to the more penetrating, longer wavelengths of light may be related to their burrowing lifestyle (Klarsfeld, 2011).

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 (morning and evening pacemaker neurons), 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. The 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).

There are at least 12 different genes encoding adenylate cyclases in the fly genome, of which the best known is Rutabaga, a calcium- and calmodulin-sensitive AC. Rut was first identified in a screen for mutations that affected learning and memory exhibited in an associative conditioning paradigm. The Rut cyclase displays the properties of a coincidence detector with its activity triggered by inputs from simultaneous activation of more than one GPCR. However, the current studies indicate that, in M pacemakers, the PDF receptor is preferentially coupled not to Rut but to the adenylate cyclase encoded by AC3. In vitro studies suggest the AC3 cyclase may be inhibited by calcium (Iourgenko, 2000). The functional consequences of this specific signaling association, the physical basis that supports it, and the degree to which it may hold true in other PDF-responsive neurons in the Drosophila brain are important questions raised by this work (Im, 2010).

The experiments that manipulated AC and PDF-R expression together indicate that relative levels of AC enzyme and receptor are important determinants of normal PDF cAMP responses in M pacemakers. Counterintuitively, AC3 over-expression was as effective in diminishing PDF responsiveness as was AC3 knockdown. One possible explanation is that the abnormally high levels of AC3 result in incorrect subcellular localization of signaling components, which may preclude the ability of AC3 to contribute to cAMP generation. Within M cells, only moderate expression of a UAS-AC3 transgene could restore normal PDF responses after knockdown of endogenous AC3. Likewise, over-expressing AC3 together with PDF-R could restore the balance between receptor and effector, as indicated by the return of PDF responsiveness. Although these results may not generalize to all cell types or receptor pathways, it is notable that, for this circadian signaling pathway, appropriate levels of signaling components were as important as their simple presence or absence. The reliance on proper stoichiometry between receptor and AC is further evidence to support the hypothesis that PDF-R and AC3 exhibit a specific functional association within the M class of pacemaker cells (Im, 2010).

One possible explanation for preferential coupling of PDF-R to AC3 is simply that it is the only adenylate cyclases to be expressed in M cells. However this explanation is inconsistent with at least two notable observations-first, M cells in flies with a severe AC3 knockdown (Df2L;GD:AC3RNAi) still elevate cAMP levels normally in response to neuropeptide DH31. Second, according to recent profiling studies, multiple other ACs are normally expressed at appreciable levels in larval LNs and in adult LN . Interestingly, these studies indicate that AC3 is not even the most abundant adenylate cyclase. Therefore, an alternative explanation is favored - that molecular specificity dictates the composition of different receptor pathways, with PDF-R residing in privileged association with AC3 (Im, 2010).

There is clear support for the concept of preferential coupling between GPCRs and specific ACs in multiple cell types, in addition to the findings in Drosophila clock cells. Previous work in Drosophila (Ueno, 2008) suggests that individual cyclases play specific roles in G-protein signaling associated with gustation. Furthermore, studies of the GABAergic system in the mouse pituitary indicate that Type 7 adenylate cyclase is associated with ethanol and CRF sensitivity, although mRNA for four of the nine mammalian ACs are detected by microarray in pituitary tissue. It has also been proposed that receptor/AC preference may depend upon environmental conditions: for example, the Type 7 preference of the CRF receptor in the mouse amygdala occurs only after phosphorylation of signaling components. Without phosphorylation, CRF receptor couples preferentially to Type 9 adenylate cyclase. Thus, the results add to the body of evidence that highly specific associations between receptors and their downstream partners are key regulators of signaling (Im, 2010).

There is clear evidence that signaling components within specific pathways do cluster, which may explain how generalized signaling molecules like cAMP and PKA are capable of targeting distinct downstream effectors. Much current work focuses on possible mechanisms for such localization, and the concept of signalosomes has been proposed to describe the spatial sequestering of signaling pathway components to promote exactly this sort of specific association. Thus preferential AC3/PDF-R coupling may be achieved by localizing AC3 near to PDF receptors. Mechanisms for grouping signaling components may include their co-localization in lipid rafts; many of the components of cAMP signaling including G proteins, PDE, PKA, and cyclic nucleotide gated channels are found in lipid rafts and studies in human bronchial smooth muscle cells detected three different AC isoforms, which are present in distinct membrane microdomains and which respond to different neurotransmitters and hormones (Im, 2010).

In addition, it is likely that another clustering mechanism includes the formation of macromolecular structures through the use of scaffolding proteins that bind to signaling molecules. Later studies showed that ACs form large complexes with β-arrestins, G proteins, and calcium channels (Davarre, 2001). The scaffolding protein InaD is required for normal localization of signaling components in the fly visual system including TRP and PLC. Specialized signaling components such as AKAPs (A-kinase anchoring proteins) can bind to receptors as well as kinases and adenylate cyclases. In Drosophila, AKAPs organize functionally discrete pools of PKA, and disruption of these signaling complexes alters normal spatio-temporal signal integration and causes a loss of anesthesia-sensitive as well as long-term olfactory memory formation in flies. In this study, knockdown of AKAP nervy reduced PDF responses: These results lead to a hypothesis whereby, in M pacemakers, PDF receptor preferentially couples to AC3 via a nervy-based scaffold system to produce normal circadian behavior. It is emphasized that, while the curren results demonstrate a functional connection between AC3 and PDF-R, the basis for any physical connections has not yet been established (Im, 2010).

Although this study provides an example of a specific receptor/enzyme pairing in a subset of circadian clock cells, the evidence also suggests the exact details of PDF signaling in other Drosophila pacemakers may differ. Simply put, the set of AC3 manipulations that caused a disruption of PDF responsiveness in M pacemakers had no such effect in E pacemakers. Importantly, disruption of Gs&α; affected both subgroups. Multiple lines of evidence have suggested that PDF signaling differs between clock cell subgroups. (1) Loss of PDF has distinct effects on PERIOD protein cycling in LNv (M cells) versus non-LNv cells (E cells). Both cell groups continued to show cycling in PER immunostaining levels and localization but, while M cells become phase-dispersed in PER cycles, E cells remain synchronized with altered phase and amplitude of PER accumulation. (2) In Pdf/cry and PDF-R/cry double mutants, a subset of E cells show a severe attenuation of the PER molecular rhythm, while M cells continue to cycle normally. Different subsets of E cells have previously been implicated in control of evening anticipation, and even when AC3 is altered in all clock cells, the evening peak retains its proper phase, again suggesting that AC3 is not a required enzyme in E type pacemaker cells. These finding are consistent with the hypothesis that there are two functionally different PDF signaling pathways. However, although this study confirmed that adenylate cyclases are responsible for the PDF FRET responses in E cells, as yet there is no positive evidence regarding the contribution of any single AC in E pacemakers. Hence it remains to be determined how uniform the components of PDF signalosomes in the M versus E pacemaker cell types are (Im, 2010).

How well do the observations obtained with neuronal imaging predict or correlate with circadian locomotor behavior? Manipulations of AC3 that severely disrupt PDF signaling in M cells were correlated with a loss of morning anticipation and increased arrythmicity in DD. Manipulations that only partially reduce the FRET response (for example, single AC3 or single nervy knockdown) resulted in normal circadian locomotor behavior or disruptions to some aspects but not all. The latter observations suggest that the animal is capable of compensating for reduced AC3-generated cAMP responses by M cells but not to complete loss of AC3 function. These data argue for a contribution to behavior by PDF signaling via AC3 in M cells and stand in contrast to a recent report by Lear (2009). That group reported that PDF-R expression in E cells alone is sufficient for morning anticipation and that exclusive expression of PDF-R in M cells does not recover morning anticipation. These differences cannot be reconciled without further experimental efforts, but it is noted that GAL80 techniques are not always sufficient to extinguish gene expression in vivo (Im, 2010).

Depending on ambient conditions, the M cells contribute to normal morning anticipatory behavior and to maintenance of rhythmicity under constant dark conditions. However, in the current study M cells expressing AC RNAi remain normally responsive to at least two other neurotransmitters (DH31 and dopamine). Hence it is suspected that much of the behavioral effect of knocking down AC3 in M pacemakers is mainly due to loss of PDF signaling in them despite retention of additional inputs from a PDF-independent source. Levels of PDF receptor and responsiveness to PDF are both high in small LNv cells and absent (or barely detectable) in large LNv cells. Therefore it is expected that AC3 alterations in M cells (directed by Pdf-GAL4) primarily affect PDF signaling in LNvs. In these considerations, the extent to which the AC3 behavioral phenotype is explained by PDF-R coupling to AC3 in M cells is not yet defined. AC3 appears coupled to at least one other GPCR pathway in LNvs because, in DD, AC3 knockdowns produced a more severe behavioral phenotype than did Pdf null flies (a higher percentage of arrhythmicity) (Im, 2010).

Knockdown of Gsα;60A levels of the M pacemakers lengthened the period in DD, a behavioral effect opposite to those seen following loss of PDF, or M cell ablation, namely. Previous studies of Gsα;60A in M cells also reported a long period phenotype. Likewise selective expression in small LNv of shibiri (a dominant negative allele of the fly homolog to dynamin or of a chronically open sodium channel both produce long period phenotypes. Although a PDF-dependent role in period lengthening cannot be ruled in the Gsα;60A experiments, imaging data suggest the lengthened period phenotype may be explained by the fact that alterations of Gsα;60A impact multiple signaling pathways (Im, 2010).

These results demonstrate in Drosophila that, in small LNv (M) circadian pacemakers, a highly specific signaling cascade is activated in response to PDF. They suggest there exists a dedicated PDF-R::AC3-dependent signaling pathway that functions to synchronize these particular clock cells. A different PDF signaling cascade is likely to operate in E pacemakers. The complete molecular details of these signaling complexes, their convergence with CRY signaling , and their ultimate connections to the cycling mechanism are significant issues for future studies (Im, 2010).

Remote control of renal physiology by the intestinal neuropeptide pigment-dispersing factor in Drosophila

The role of the central neuropeptide pigment-dispersing factor (PDF) in circadian timekeeping in Drosophila is remarkably similar to that of vasoactive intestinal peptide (VIP) in mammals. Like VIP, PDF is expressed outside the circadian network by neurons innervating the gut, but the function and mode of action of this PDF have not been characterized. This study investigated the visceral roles of PDF by adapting cellular and physiological methods to the study of visceral responses to PDF signaling in wild-type and mutant genetic backgrounds. Intestinal PDF acts at a distance on the renal system, where it regulates ureter contractions. PdfR, PDF's established receptor, is expressed by the muscles of the excretory system, and evidence is presented that PdfR-induced cAMP increases underlie the myotropic effects of PDF. These findings extend the similarities between PDF and VIP beyond their shared central role as circadian regulators, and uncover an unexpected endocrine mode of myotropic action for an intestinal neuropeptide on the renal system (Talsma, 2012).

Striking similarities have been found between fly PDF and mammalian VIP in the generation of daily behavioral rhythms. Outside the central clock, mammalian VIP and its related ligand PACAP (pituitary adenylate cyclase activating peptide) act on smooth-muscle receptors in a variety of internal organs, including those of the lower urinary tract (Talsma, 2012).

The findings of broad visceral PdfR expression and a function for PDF signaling in the regulation of visceral muscle contraction extend the similarities between these peptides and their receptors beyond the central clock. The PdfR-expressing circular muscles of the ureter control the flow of urine into the terminal portion of the digestive tract in a manner analogous to the VPAC2R+ detrusor muscle of the mammalian bladder. Abnormal fluid regulation has also been described in mice lacking VIP or PACAP, so it will be of interest to establish if PDF signaling has related in vivo roles in flies. The presence of VIP/PACAP immunoreactivity in neural fibers of the lower urinary tract of mammals suggests a local, transmitter-like action on the excretory muscle receptors, and VIP is generally believed to act as a local neurotransmitter rather than as a circulating hormone. However, systemic effects of gut-derived VIP cannot be ruled out. Indeed, VIP plasma levels increase after an oral osmotic load or intravenous application of cholinesterase inhibitors. In light of the current findings, which reveal a role for circulating PDF on the renal system, a possible systemic function of VIP deserves further investigation using, for example, transgenic mice lacking the peptide in specific (enteric vs. renal) neuronal populations (Talsma, 2012).

Previous work has made use of the larger viscera of locusts or crickets to establish that some neuropeptides induce renal tubule movements. The results using Drosophila indicate that renal regulators are secreted from an unexpected source, the gut-innervating ab-PDF neurons, to act as endocrine regulators of the renal muscles, which are entirely devoid of innervation. Indeed, dTrpA1 experiments strongly suggest that the normal ligand for visceral PdfR is released from the gut-innervating ab-PDF neurons, because the only other site of PDF production in mature adults are the clock interneurons in the brain, which are thought to be chemically insulated from the hemolymph by the blood-brain barrier and were not present during dTrpA1 excitation experiments. Consistent with this idea, extracts of the abdominal ganglia of another insect, Carausius morosus, have a potent stimulatory action on renal tubule writhing, the active factors in which were suspected to be peptide (Talsma, 2012).

The directionality of this endocrine signal from the gut to the renal system is unexpected and may be of relevance to mammals, where functional links between the digestive and excretory systems are well established with regard to absorption and secretion, and evidence suggests that communication between the two systems might facilitate their concerted action. An enterorenal axis, whereby unidentified gut-derived signals affect kidney function directly, has recently been proposed on the basis of two observations: the activity of duodenal mucosa homogenates on kidney ion secretion and the differential effect of intravenously versus orally administered sodium loads on renal sodium excretion. In summary, the current findings indicate a role for PDF in the control of visceral physiology in the fly, thereby extending the similarities between fly PDF and mammalian VIP beyond their shared role in circadian timekeeping. These findings have also revealed a peptidergic enterorenal axis in Drosophila, the evolutionary conservation and significance of which deserves further investigation (Talsma, 2012).

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. 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. The 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).

EFFECTS OF MUTATION pdf-null animals were discovered among laboratory stocks as a fortuitous consequence of studying ectopic P element reporter gene expression. The pWF6-84 stock contains a dFMRF-lacZ fusion gene that produces ectopic reporter expression in a pattern that includes the LN_v neurons. pWF6-84 was mobilized and several lines were found that lacked all anti-PAP immunostaining and lacked beta-PDH immunostaining in most beta-PDH-positive cells. The lack of PAP immunostaining was heritable and specific for the PDF transmitter system. W33 and W15 lines were both positive when stained with other anti-neuropeptide antibodies (proFMRF). In W15 animals, beta-PDH signals were absent in LN_v, and in PDF-Tri and PDF-Ab neurons. However, signals were retained in neurons of the pars intercerebralis (PI) and protocerebrum (PL). PI neurons are present in both W33 and W15 flies. Neurons lacking beta-PDH signals in W15 correspond to those that express PDF mRNA and PDF in wild-type; this supports the hypothesis that PI and PL neurons do not express the Pdf gene product but instead express cross-reacting PDF related-antigen(s). The pdf gene from W15 animals was sequenced and a nonsense mutation was found at prepro-PDF residue 21, converting a Tyr to a stop codon. This mutation is referred to as pdf⁰¹. The conceptual pdf⁰¹ precursor is consistent with the immunostaining phenotype because it is truncated before epitope positions assayed by anti-beta-PDH, PDF, or PAP antibodies. With a PCR-based assay, the pdf⁰¹ mutation was found in each of the 22 derivative lines that displayed the phenotype. The same assay revealed that pdf⁰¹ is present in various laboratory stocks at a range of frequencies: some are homozygous for the allele. In a survey of 38 y w^67c23 adults, the stock into which the pWF6-84 DNA was injected, 9 flies homozygous and 20 heterozygous for pdf⁰¹ were found (Renn, 1999).

The lack of peptide expression could result from either the absence of PDF neurons or merely a lack of peptide transmitter expression in otherwise normal neurons. To determine the state of pdf neurons in animals containing the transmitter mutation, pdf-GAL4 was used to produce beta-galactosidase (beta-gal) in flies that contained a single pdf⁺ allele or that were hemizygous for pdf⁰¹. In all pdf⁰¹/deletion specimens, the number and morphology of LN_v, PDF-Tri, and PDF-Ab neurons were normal (Renn, 1999).

Over the course of 24 hr in light:dark (LD) cycles, wild-type flies are active at dawn, quieter at midday, then active again toward evening. One feature of flies entrained to LD cycling is the anticipation of transitions between lights-on and -off. Rhythmic behavior persists when wild-type flies proceed from LD into constant darkness. The locomotor activity rhythms of the pdf⁰¹ mutant were examined. Homozygous and hemizygous pdf⁰¹ flies are well entrained during LD cycles. However, pdf⁰¹ behavior in LD is not entirely normal. The evening activity peak is advanced by approximately 1 hr. Also, there is a lack of lights-on anticipation. Free-running behavior of pdf⁰¹ in constant darkness (DD) is severely abnormal and includes several features that distinguish this mutation from others that disrupt circadian behavior. By periodogram analysis, flies homozygous and hemizygous for pdf⁰¹ are much less rhythmic in DD than are pdf⁺ controls. Of these mutants, 50%-98% exhibited no detectable rhythmicity for the duration of 9 DD days. Actograms of pdf⁰¹ individuals suggest that most are rhythmic for 2 or 3 days in DD but later lose rhythmicity. Separate average activity histograms for DD days 1-2 and DD days 3-9 reveal the severity of the pdf⁰¹ phenotype. A higher proportion of mutant individuals are arrhythmic during DD days 3-9 than during DD days 1-9. The minority of pdf⁰¹ animals that maintain DD rhythmicity have free-running periods approximately 1 hr shorter than wild-type or pdf⁰¹/+ heterozygous flies (Renn, 1999).

Signal-to-noise analysis (SNR) was used to obtain measures that are linearly related to rhythm strength. SNR stems from Maximum Entropy Spectral Analysis (MESA) of the locomotor records: it provides single values for each fly's behavior and permits quantitation of weak rhythmicity, such as displayed by pdf⁰¹ flies. The average SNR for pdf⁺ flies depends on the background genotype and ranges from 1.7 for wild type to 1.0 for y w. Both stocks display normal proportions of rhythmic individuals and periods. The range of SNR values associated with the behavior of wild-type flies is very wide (0.4-4). In contrast, the distributions for the behaviorally arrhythmic per⁰¹ and disco mutants are skewed to the left, as expected. The distributions of per⁰¹ SNRs for DD days 1-9 and those for the DD days 3-9 are congruent. This reflects the fact that this period mutant is arrhythmic throughout the DD period. SNRs for pdf⁰¹ mutant animals are similar to those of per⁰¹, even when computed for the entire DD 1-9. Hence, the residual rhythms displayed by pdf⁰¹ animals in DD are very weak (Renn, 1999).

In Drosophila, the amidated neuropeptide Pigment dispersing factor (PDF) is expressed by the ventral subset of lateral pacemaker neurons and is required for circadian locomotor rhythms. Residual rhythmicity in pdf mutants likely reflects the activity of other neurotransmitters. It was asked whether other neuropeptides contribute to such auxiliary mechanisms. The gal4/UAS system was used to create mosaics for the neuropeptide amidating enzyme PHM; amidation is a highly specific and widespread modification of secretory peptides in Drosophila. Three different gal4 drivers restrict PHM expression to different numbers of peptidergic neurons. These mosaics display aberrant locomotor rhythms to degrees that parallel the apparent complexity of the spatial patterns. Certain PHM mosaics are less rhythmic than pdf mutants and as severe as per mutants. Additional gal4 elements were added to the weakly rhythmic PHM mosaics. Although adding pdf-gal4 provided only partial improvement, adding the widely expressed tim-gal4 largely restored rhythmicity. These results indicate that, in Drosophila, peptide amidation is required for neuropeptide regulation of behavior. They also support the hypothesis that multiple amidated neuropeptides, acting upstream, downstream, or in parallel to PDF, help organize daily locomotor rhythms (Taghert, 2001).

Identification of genes involved in Drosophila melanogaster geotaxis, a complex behavioral trait

Identifying the genes involved in polygenic traits has been difficult. In the 1950s and 1960s, laboratory selection experiments for extreme geotaxic behavior in fruit flies established for the first time that a complex behavioral trait has a genetic basis. But the specific genes responsible for the behavior have never been identified using this classical model. To identify the individual genes involved in geotaxic response, cDNA microarrays were used to identify candidate genes and fly lines mutant in these genes were assessed for behavioral confirmation. The identities of several genes that contribute to the complex, polygenic behavior of geotaxis have thus been determined (Toma, 2002).

Pioneering experiments on Drosophila melanogaster and Drosophila pseudoobscura investigated the nature of the genetic basis for extreme, selected geotaxic behavior. These experiments constituted the first attempt at the genetic analysis of a behavior. Selection and chromosomal substitution experiments successfully showed that there is a genetic basis for extreme geotaxic response in flies and, by implication, for behavior in general. These experiments also added to understanding of the role of variation in phenotypic evolution and selection. Despite their seminal contributions in behavioral genetics, population genetics and the study of selection, by their nature these experiments could not identify specific genes (Toma, 2002 and references therein).

These results highlight both the success and the limitation of behavioral selection experiments. Although selection results tend to be representative of the natural interactions of genes that produce behavior and can demonstrate that a trait has a genetic basis, they do not pinpoint specific genes that influence the trait. This is partly due to the involvement of many genes and the relatively minor role of each in complex polygenic phenotypes -- a problem that is especially acute for the intrinsically more variable phenotypes that are associated with behavior. The advent of cDNA microarray technology offers an easily generalized strategy for detecting gene expression differences and can complement other means of identifying the genes that underlie complex traits. An expression difference may occur in a gene that is not itself polymorphic, but that gene may contribute to the realization of the phenotypic difference (Toma, 2002).

As a starting point for identifying genes that affect a complex trait, the selected, established Hi5 and Lo extreme geotaxic lines were examined for changes in gene expression between strains of Drosophila melanogaster subjected to long-term selection and isolation. A two-step approach was used: (1) the differential expression levels of mRNAs isolated from the heads of Hi5 and Lo flies was determined using cDNA microarrays and real-time quantitative PCR (qPCR); (2) a subset of the differentially expressed genes was independently tested for their influence on geotaxis behavior by running mutants for these genes through a geotaxis maze. It was reasoned that some of the differences in gene expression between strains might be related to phenotypic differences and that it should therefore be possible, at least in part, to reconstruct the phenotype with independently derived mutations in some of the differentially expressed genes (Toma, 2002).

The findings indicate that differences in gene expression can be used to identify phenotypically relevant genes, even when no large, single-gene effects are detectable by classical, quantitative genetic analysis. Three of the four genes implicated by microarray and qPCR measurements caused differences in geotaxis, whereas none of the six control genes had an effect. Only those genes that had larger differences in expression according to the microarrays, or that were significantly different according to qPCR results (cry, Pdf and Pen), significantly changed geotaxis scores. The converse was not true, because altered geotaxis behavior did not always accompany larger differences in mRNA levels, as shown by pros, although this might reflect the sensitivity of pros to aspects of the genetic context. All of the genes tested for which there was little or no difference in mRNA levels between the selected Hi5 and Lo lines also showed no influence on geotaxis behavior (Toma, 2002).

The directionality of behavioral and mRNA differences proved to be consistent with predictions that were based on expression levels. Homozygous null mutants of Pdf and cry showed a significant increase in geotaxis score, which is consistent with a lower level of expression of these genes in Hi5 relative to Lo. Similarly, the heterozygous Pen mutant showed a significant downward shift in geotaxis score, which is consistent with a lower level of Pen expression in Lo relative to Hi5. Thus, the change in behavior of the tested mutants corresponds to the direction predicted by differences in transcript level in the selected Hi5 and Lo lines (Toma, 2002).

Whereas the cry, Pen and female Pdf mutants produced the anticipated effect on behavior, the magnitude of behavioral effect was smaller than in the original selected lines. This probably reflects the difference between the aggregate effect of an ensemble of genes in the selected lines as opposed to the individual effect of a single mutant gene in a neutral background. In addition, their relatively small effects are exactly the results that one would predict in a polygenic system such as geotaxis behavior, in which many genes have small contributions to the overall phenotype. The three genes identified in this study would not have been predicted on the basis of their previously defined functions (Toma, 2002).

These results show that the two separate approaches to behavioral genetics -- the classical Hirschian quantitative analysis of genetic architecture and the modern Benzerian approach of single-gene mutant analysis -- are complementary and can be unified. This study used the results of a Hirschian approach of laboratory selection for natural variants to identify single gene differences, such as one would find in a Benzerian approach. The results are consistent with the suggestion that naturally occurring variants in behavior correspond to mild lesions in pleiotropic genes (Toma, 2002).

Finally, the results show that differences in gene expression identified by cDNA microarray analysis can be used as a starting point for narrowing down the numbers of candidate genes involved in complex genetic processes. Such an approach is analogous, as well as complementary, to the current method of mapping quantitative trait loci to large chromosomal intervals and then making educated guesses about which genes within those intervals may be involved in the trait (Toma, 2002).

The combination of selection, with its ability to exaggerate natural phenotypic variation, and global analysis of differences in gene expression by cDNA microarray analysis offers a promising approach to previously intractable molecular analyses of behavior. The geotaxis genes that were identified might have been the direct targets of selection, or they might be downstream of the direct targets. Additional studies using the Hi5 and Lo selected lines will be required to distinguish between these possibilities and to determine the causal role that these genes have in the context of the selected lines (Toma, 2002).

This study has gone from the selection of a 'laboratory-evolved' behavioral phenotype, to screening for mRNA differences, to partially reconstituting the phenotype using mutants. This shows the feasibility of combining genomic and classical genetic approaches for the breakdown and partial reassembly of an artificially selected behavioral trait (Toma, 2002).

Drosophila free-running rhythms require intercellular communication: Persistent molecular rhythm requires Pigment-dispersing factor

Robust self-sustained oscillations are a ubiquitous characteristic of circadian rhythms. These include Drosophila locomotor activity rhythms, which persist for weeks in constant darkness (DD). Yet the molecular oscillations that underlie circadian rhythms damp rapidly in many Drosophila tissues. Although much progress has been made in understanding the biochemical and cellular basis of circadian rhythms, the mechanisms that underlie the differences between damped and self-sustaining oscillations remain largely unknown. A small cluster of neurons in adult Drosophila brain, the ventral lateral neurons (LN(v)s), is essential for self-sustained behavioral rhythms and has been proposed to be the primary pacemaker for locomotor activity rhythms. With an LN(v)-specific driver, functional clocks were restricted to these neurons and it was shown that they are not sufficient to drive circadian locomotor activity rhythms. Also contrary to expectation, it was found that all brain clock neurons manifest robust circadian oscillations of timeless and cryptochrome RNA for many days in DD. This persistent molecular rhythm requires Pigment-dispersing factor (PDF), an LN(v)-specific neuropeptide, because the molecular oscillations are gradually lost when Pdf⁰¹ mutant flies are exposed to free-running conditions. This observation precisely parallels the previously reported effect on behavioral rhythms of the Pdf⁰¹ mutant. PDF is likely to affect some clock neurons directly, since the peptide appears to bind to the surface of many clock neurons, including the LN(v)s themselves. The brain circadian clock in Drosophila is clearly distinguishable from the eyes and other rapidly damping peripheral tissues, since it sustains robust molecular oscillations in DD. At the same time, different clock neurons are likely to work cooperatively within the brain, because the LN(v)s alone are insufficient to support the circadian program. Based on the damping results with Pdf⁰¹ mutant flies, it is proposed that LN(v)s, and specifically the PDF neuropeptide that it synthesizes, are important in coordinating a circadian cellular network within the brain. The cooperative function of this network appears to be necessary for maintaining robust molecular oscillations in DD and is the basis of sustained circadian locomotor activity rhythms (Peng, 2003).

The neuropeptide pigment-dispersing factor coordinates pacemaker interactions in the Drosophila circadian system

In Drosophila, the neuropeptide pigment-dispersing factor (PDF) is required to maintain behavioral rhythms under constant conditions. To understand how PDF exerts its influence, time-series immunostainings were performed for the Period protein in normal and pdf mutant flies over 9 d of constant conditions. Without pdf, pacemaker neurons that normally express PDF maintained two markers of rhythms: that of Period nuclear translocation and its protein staining intensity. As a group, however, they displayed a gradual dispersion in their phasing of nuclear translocation. A separate group of non-PDF circadian pacemakers also maintained Period nuclear translocation rhythms without pdf but exhibit altered phase and amplitude of Period staining intensity. Therefore, pdf is not required to maintain circadian protein oscillations under constant conditions; however, it is required to coordinate the phase and amplitude of such rhythms among the diverse pacemakers. These observations begin to outline the hierarchy of circadian pacemaker circuitry in the Drosophila brain (Lin, 2004).

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).

Circadian pacemaker neurons change synaptic contacts across the day

Daily cycles of rest and activity are a common example of circadian control of physiology. In Drosophila, rhythmic locomotor cycles rely on the activity of 150-200 neurons grouped in seven clusters. Work from many laboratories points to the small ventral lateral neurons (sLNvs) as essential for circadian control of locomotor rhythmicity. sLNv neurons undergo circadian remodeling of their axonal projections, opening the possibility for a circadian control of connectivity of these relevant circadian pacemakers. This study shows that circadian plasticity of the sLNv axonal projections has further implications than mere structural changes. First, it was found that the degree of daily structural plasticity exceeds that originally described, underscoring that changes in the degree of fasciculation as well as extension or pruning of axonal terminals could be involved. Interestingly, the quantity of active zones changes along the day, lending support to the attractive hypothesis that new synapses are formed while others are dismantled between late night and the following morning. More remarkably, taking full advantage of the GFP reconstitution across synaptic partners (GRASP) technique, this study showed that, in addition to new synapses being added or removed, sLNv neurons contact different synaptic partners at different times along the day. These results lead to a proposal that the circadian network, and in particular the sLNv neurons, orchestrates some of the physiological and behavioral differences between day and night by changing the path through which information travels (Gorostaza, 2014).

Circadian remodeling of the small ventral lateral neuron (sLNv) dorsal terminals was first described at the peak and trough levels of pigment-dispersing factor (PDF) immunoreactivity, that is at zeitgeber time 2 (ZT2) and ZT14 (2 hr after lights ON and lights OFF, respectively), as well as their counterparts under constant darkness (DD) (circadian time 2 [CT2] and CT14). For a more precise examination of the extent of structural remodeling, a time course was carried out. An inducible GAL4 version termed GeneSwitch restricted to PDF neurons (pdf-GS) combined with a membrane-tethered version of GFP (mCD8GFP) was used as control. As expected from the original observations, a significant reduction in complexity of the axonal arbor-measured as total axonal crosses-could be seen between CT2 and CT14 and between CT18 and CT22, which remained unchanged at nighttime. However, toward the end of the subjective night (CT22), the primary processes appeared to be shorter. To more precisely describe this additional form of plasticity, the length of the maximum projection was measure from the lateral horn toward the midbrain. This analysis revealed that toward the end of the subjective night (CT22), PDF projections are significantly shorter than at the beginning of the day (CT2). These observations imply that mechanisms other than the proposed changes in the degree of fasciculation are recruited during circadian plasticity. To get a deeper insight into the nature of the phenomena, the changes were monitored in brain explants kept in culture for 48 hr after dissection. Transgenic pdf-GAL4; UAS-mCD8RFP flies (referred to as pdf>RFP) were dissected under safe red light, and brains were maintained under DD. Imaging of individual brains at two different time points highlighted three types of changes experienced by axonal terminals: (1) changes in the degree of fasciculation/defasciculation, more common in primary branches, (2) the addition/retraction of new processes, mostly affecting those of secondary or tertiary order, and (3) positional changes of minor terminals, thus confirming and extending previous observations. Altogether, these results indicate that a rather complex remodeling process takes place on daily basis in the axonal terminals of PDF neurons (Gorostaza, 2014).

The level of structural remodeling occurring at the dorsal terminals suggested that synapses themselves could undergo changes in a time-dependent fashion. The presynaptic protein Synaptotagmin (SYT) was examined at different times across the day as an indicator of vesicle accumulation. A GFP-tagged version of SYT was expressed in PDF neurons (pdf >syt^GFP), and both the number and area span by SYT+ puncta (most likely describing the accumulation of several dense core vesicles) were analyzed separately at the sLNv dorsal terminals. No statistical differences were observed in the number of SYT+ puncta (although there is a tendency for higher numbers in the early morning), perhaps as a result of the nature of the signal, which is too diffuse for precise identification of individual spots. On the other hand, SYT+ puncta were larger and, as a result, the area covered by SYT+ immunoreactivity was significantly different at CT2 compared to CT14, but not between CT22 and CT2, perhaps reflecting that vesicles started to accumulate at the end of the day in preparation for the most dramatic membrane change taking place between CT22 and the beginning of the following morning (Gorostaza, 2014).

The observation that a more complex structure correlated with a larger area covered by presynaptic vesicles reinforced the notion that indeed the number of synapses could be changing throughout the day and prompted analysis of Bruchpilot (BRP), a well-established indicator of active zones. Expressing a tagged version of BRP in PDF neurons, the number of BRP+ puncta was quantitated as a proxy for active zones at times when the most dramatic changes in structure had been detected (i.e., CT2, CT14, and CT22). Interestingly, the number of active zones was significantly larger at CT2 than at CT14 or CT22; in fact, no statistical differences were observed between the last two time points, underscoring that axonal remodeling can occur (i.e., pruning of major projections taking place toward the end of the night) without significantly affecting overall connectivity. Thus, circadian structural plasticity is accompanied by changes in the number of synapses. Not only are more vesicles recruited toward CT2, but also a higher number of active zones are being established (Gorostaza, 2014).

Circadian changes in the abundance of the presynaptic active zone BRP have also been shown in the first optic neuropil of the fly brain, although BRP abundance in the lamina increases in the early night under DD conditions, in contrast to the oscillations in BRP levels observed at the dorsal protocerebrum that peak in the early subjective day just described. In addition, rhythmic changes in the number of synapses have also been described in the terminals of adult motor neurons in Drosophila examined through transmission electron microscopy, as well as BRP+ light confocal microscopy, underscoring the validity of the approach employed herein. Interestingly, in different brain areas, the level of presynaptic markers (such as BRP^RFP or SYT^GFP) also changes in response to the sleep/wake 'state,' being high when the animals are awake and lower during sleep; this observation led to the proposal that sleep could be involved in maintaining synaptic homeostasis altered during the awaking state. This trend coincides with observation of higher levels during the subjective morning and lower levels at the beginning of the subjective night; however, no changes were detected through the night, suggesting that, at least in clock neurons, there is a circadian rather than a homeostatic control of synaptic activity. Given that clock outputs are predominantly regulated at the transcriptional level and that there is circadian regulation of MEF2, a transcription factor that turns on a program involved in structural remodeling, this correlation opens the provocative possibility that the circadian clock is controlling the ability of assembling novel synapses in particularly plastic neurons, which might become recruited and/or stabilized, or otherwise pruned (disassembled), toward the end of the day (Gorostaza, 2014).

Adult-specific electrical silencing of PDF neurons reduces the complexity of dorsal arborizations, although a certain degree of circadian remodeling of the axonal terminals still takes place. To examine whether electrical alterations could affect circadian changes in the number of active zones, either Kir2.1 or NaChBac was expressed (to hyperpolarize or depolarize PDF neurons, respectively). To avoid any undesired developmental defects, pdf-GS was used to drive expression of the channels only during adulthood. Interestingly, Kir2.1 expression abrogated circadian changes in the number of active zones. In fact, PDF neurons displayed a reduced number of active zones compared to controls at CT2 and remained at similar levels throughout the day, indistinguishable from nighttime controls. On the other hand, when neurons were depolarized through NaChBac expression, the number of active zones did not change along the day and was maintained at daytime levels even at CT14 and CT22 (Gorostaza, 2014).

It has recently been shown that MEF2, a transcription factor involved in activity-dependent neuronal plasticity and morphology in mammals, is circadianly regulated and mediates some of the remodeling of PDF dorsal terminals through the regulation of Fasciclin2. In contrast, adult-specific silencing (and depolarization) of PDF neurons abolishes cycling in the number of BRP+ active zones, despite the fact that it does not completely obliterate the remodeling of the axonal terminals, suggesting that some of the mechanisms underlying structural plasticity are clearly activity independent and are most likely the result of additional clock-controlled output pathways still to be identified (Gorostaza, 2014).

Since structural remodeling of PDF neurons results in the formation and disappearance of new synapses on daily basis, it was anticipated that not only the number but also the postsynaptic partners of these contacts could concomitantly be changing. To shed light on this possibility, GFP reconstitution across synaptic partners (GRASP), which labels contacts between adjacent membranes, was used. In brief, two complementary fragments of GFP tethered to the membrane are expressed in different cells. If those cells are in contact, GFP is reconstituted and becomes fluorescent. GRASP has previously been employed to monitor synapses in adult flies. Given the complex arborization at the dorsal protocerebrum, it was asked whether specific subsets of circadian neurons projecting toward that area could be contacting across the day. Perhaps not surprisingly, an extensive reconstituted GFP signal could be observed between the sLNv dorsal projections and those of the posterior dorsal neuron 1 cells (DN1ps, lighted up by the dClk4.1-GAL4 line, suggesting contacts along the entire area, which are detectable across all time points analyzed (ZT2, ZT14, and ZT22). Consistent with these observations, extensive physical contact between the sLNv projections and those of the DN1p neurons has just been reported at the dorsal protocerebrum with no clear indication of the time of day examined. Next the study examined whether a subset of dorsal LNs (LNds), projecting toward both the accessory medulla and the dorsal protocerebrum (through the combined expression of Mai179-GAL4; pdf-GAL80), could also contact the profuse dorsal arborization of sLNv neurons; this genetic combination enables expression of split-GFP in a restricted number of circadian cells (which are part of the evening oscillator, i.e., up to four LNds, including at least a CRYPTOCHROME-positive one, and the fifth sLNv), as well as others located within the pars intercerebralis (PI), a neurosecretory structure recently identified as part of the output pathway relevant in the control of locomotor behavior. In contrast to the extensive connections between DN1p and sLNv clusters, only very discreet reconstituted puncta were detected. Quite strikingly, the degree of connectivity appeared to change across the day, reaching a maximum (when almost every brain exhibited reconstituted signal) at ZT22. However, due to the nature of the signal, no quantitation of its intensity was attempted. Although a more detailed analysis is required to define the identity (i.e., whether it is one or several LNds, the fifth sLNv, or both groups that directly contact the sLNvs), this finding highlights a potentially direct contact between the neuronal substrates of the morning and evening oscillators. In sum, through GRASP analysis, this study has begun to map the connectivity within the circadian network; commensurate with a hierarchical role, the sLNvs appear to differentially contact specific subsets in a distinctive fashion (Gorostaza, 2014).

To address the possibility that PDF neurons could be contacting noncircadian targets at different times across the day, an enhancer trap screen was carried out employing a subset of GAL4 enhancers selected on the basis of their expression pattern in the adult brain, i.e., known to drive expression in the dorsal protocerebrum, and an additional requirement imposed was that none of the selected GAL4 lines could direct expression to the sLNv neurons to avoid internal GFP reconstitution. Reconstitution of the GFP signal at the sLNv dorsal terminals by recognition through specific antibodies was assessed at three different time points for each independent GAL4 line (ZT2, ZT14, and ZT22). Some of the GAL4 lines showed reconstituted GFP signal at every time point analyzed, suggesting that those neuronal projections are indeed in close contact across the day and might represent stable synaptic contacts. No GFP signal was detected in the negative parental controls. Despite the fact that several GAL4 drivers directed expression to the proximity of the PDF dorsal terminals, some of the selected lines did not result in reconstituted GFP signal (Gorostaza, 2014).

Quite remarkably, a proportion of the GAL4 lines showed GFP+ signal only at a specific time point. One such example is line 3-86, where reconstitution was detected in most of the brains analyzed at ZT2, but not at nighttime. Being able to identify putative postsynaptic contacts to the sLNvs in the early morning is consistent with the observation of a higher number of BRP+ active zones in the early day. This enhancer trap spans different neuropils, such as the mushroom body (MB) lobes and lateral horn, and directs expression to particularly high levels in the PI, a structure that has recently been implicated in the rhythmic control of locomotor activity. In fact, some yet unidentified somas in the PI appear to arborize profusely near the PDF dorsal terminals, underscoring a potential link between the two neuronal groups. These direct contacts are unlikely to be the ones reported by Mai179-GAL4; pdf-GAL80 since those connect to the sLNv neurons preferentially at night. Interestingly, a subset of neurons in the PI is relevant in mediating the arousal promoting signal from octopamine; in addition, sleep promoting signals are also derived from a different subset of neurons in the PI, opening the attractive possibility that both centers could be under circadian modulation (Gorostaza, 2014).

GRASP analysis also uncovered a different neuronal cluster (4-59) that contacts PDF neurons preferentially during the early night (ZT14), which is in itself striking, since this time point corresponds to that with fewer arborizations and an overall decrease in the number of synapses. This enhancer trap is expressed in the MBs, subesophagic ganglion, antennal lobes, and accessory medulla. Among those structures, the MBs are important for higher-order sensory integration and learning in insects. Interestingly, circadian modulation of short-term memory and memory retrieval after sleep deprivation has been reported; short-term memory was found to peak around ZT15-ZT17, coinciding with the window of GFP reconstitution, thus providing a functional connection to the synaptic plasticity observed. To corroborate whether there is a direct contact between the two neuronal clusters, the extensively used GAL4 driver OK107, which is expressed in the α'/β'and the γ lobes of the MBs and to a lower extent in the PI, was employed for GRASP analysis. Surprisingly, reconstituted GFP signal could be observed at every time point analyzed, suggesting that MB lobes contact PDF neurons throughout the day but that specific clusters (for example those highlighted by the 4-59 line) establish plastic, time-of-day-dependent physical contact with PDF neurons (Gorostaza, 2014).

It was next asked whether these prospective postsynaptic targets of PDF neurons could play a role in the output pathway controlling rhythmic locomotor activity. To address this possibility, the impact of adult-specific alteration of excitability of distinct neuronal groups was examined through expression of TRPA1. Interestingly, adult-specific depolarization of specific neuronal populations triggered a clear deconsolidation of the rhythmic pattern of activity, which resulted in less-rhythmic flies accompanied by a significant decrease in the strength of the underlying rhythm. These results lend support to the notion that the underlying neuronal clusters are relevant in the control of rest/activity cycles (Gorostaza, 2014).

Over the years, it has become increasingly clear that the circadian clock modulates structural properties of different cells. In fact, a number of years ago, it was reported that the projections of a subset of core pacemaker fly PDF+ and mammalian VIP+ neurons undergo structural remodeling on daily basis. The work presented in this study lends support to the original hypothesis that circadian plasticity represents a means of encoding time-of-day information. By changing their connectivity, PDF neurons could drive time-specific physiological processes. As new synapses assemble while others are dismantled, the information flux changes, allowing PDF neurons to promote or inhibit different processes at the same time. This type of plasticity adds a new level to the complex information encoded in neural circuits, where PDF neurons could not only modulate the strength in the connectivity between different partners, but also define which neuronal groups could be part of the circadian network along the day. Although further analysis of the underlying process is ensured, evidence so far supports the claim that structural plasticity is an important circadian output (Gorostaza, 2014).

Ultradian rhythm unmasked in the Pdf clock mutant of Drosophila

A diverse range of organisms shows physiological and behavioural rhythms with various periods. Extensive studies have been performed to elucidate the molecular mechanisms of circadian rhythms with an approximately 24 h period in both Drosophila and mammals, while less attention has been paid to ultradian rhythms with shorter periods. This study used a video-tracking method to monitor the movement of single flies, and clear ultradian rhythms were detected in the locomotor behaviour of wild type and clock mutant flies kept under constant dark conditions. In particular, a Pigment-dispersing factor mutant demonstrated a precise and robust ultradian rhythmicity, which was not temperature compensated. These results suggest that Drosophila has an endogenous ultradian oscillator that is masked by circadian rhythmic behaviours (Seki, 2014).

Synchronous Drosophila circadian pacemakers display nonsynchronous Ca²⁺ rhythms in vivo

In Drosophila, molecular clocks control circadian rhythmic behavior through a network of ~150 pacemaker neurons. To explain how the network's neuronal properties encode time, this study performed brainwide calcium imaging of groups of pacemaker neurons in vivo for 24 hours. Pacemakers exhibit daily rhythmic changes in intracellular Ca(2+) that are entrained by environmental cues and timed by molecular clocks. However, these rhythms are not synchronous, as each group exhibits its own phase of activation. Ca(2+) rhythms displayed by pacemaker groups that are associated with the morning or evening locomotor activities occur ~4 hours before their respective behaviors. Loss of the receptor for the neuropeptide PDF promotes synchrony of Ca(2+) waves. Thus, neuropeptide modulation is required to sequentially time outputs from a network of synchronous molecular pacemakers (Liang, 2016). behaviors induced by geomagnetic field in Drosophila

Appropriate vertical movement is critical for the survival of flying animals. Although negative geotaxis (moving away from Earth) driven by gravity has been extensively studied, much less is understood concerning a static regulatory mechanism for inducing positive geotaxis (moving toward Earth). Using Drosophila melanogaster as a model organism, this study showed that geomagnetic field (GMF) induces positive geotaxis and antagonizes negative gravitaxis. Remarkably, GMF acts as a sensory cue for an appetite-driven associative learning behavior through the GMF-induced positive geotaxis. This GMF-induced positive geotaxis requires the three geotaxis genes, such as cry, the cation channel pyx and pdf, and the corresponding neurons residing in Johnston's organ of the fly's antennae. These findings provide a novel concept with the neurogenetic basis on the regulation of vertical movement by GMF in the flying animals (Bae, 2016).

EVOLUTIONARY HOMOLOGS

Serotonin and the Neuropeptide PDF Initiate and Extend Opposing Behavioral States in C. elegans

Foraging animals have distinct exploration and exploitation behaviors that are organized into discrete behavioral states. This study characterized a neuromodulatory circuit that generates long-lasting roaming and dwelling states in Caenorhabditis elegans. Two opposing neuromodulators, serotonin and the neuropeptide pigment dispersing factor (PDF), each initiate and extend one behavioral state. Serotonin promotes dwelling states through the MOD-1 serotonin-gated chloride channel. The spontaneous activity of serotonergic neurons correlates with dwelling behavior, and optogenetic modulation of the critical MOD-1-expressing targets induces prolonged dwelling states. PDF promotes roaming states through a Gαs-coupled PDF receptor; optogenetic activation of cAMP production in PDF receptor-expressing cells induces prolonged roaming states. The neurons that produce and respond to each neuromodulator form a distributed circuit orthogonal to the classical wiring diagram, with several essential neurons that express each molecule. The slow temporal dynamics of this neuromodulatory circuit supplement fast motor circuits to organize long-lasting behavioral states (Flavell, 2013).

These results show that serotonergic signaling through mod-1 initiates and extends dwelling states, whereas PDF signaling through pdfr-1 initiates and extends roaming states. What does a complete neuromodulatory circuit look like in its organization and its relationship to fast circuits? Despite the compact size of the C. elegans nervous system, the serotonin and PDF that regulate roaming and dwelling each have several important sources, and their receptors each act in several target neurons. These modulatory circuits include sensory neurons, interneurons, and motor neurons, but their organization does not follow the dominant sensory-to-motor hierarchy of the classical synaptic wiring diagram. The serotonin sources, NSM and HSN, are both motor neurons (although NSM may have sensory functions as well), and the PDF sources are interneurons; the targets include sensory neurons (ASI) and multiple interneurons. The serotonin and PDF circuits are mostly nonoverlapping, intersecting only at the AIY neurons. Moreover, although serotonin- and PDF-expressing neurons have chemical and electrical synapses in the C. elegans wiring diagram, these synapses do not overlap with the neuromodulatory connections inferred from the genetic mapping experiments. Thus, the neuromodulatory circuit for long-lasting behavioral states is essentially orthogonal to the synaptic connectivity diagram. Extrasynaptic function of neuromodulators is well established in C. elegans and other animals, but this need not imply that serotonin and PDF act as systemic hormones. All of the relevant neurons in this neuromodulatory circuit have processes in or near the C. elegans nerve ring, suggesting that diffusion over ∼100-200 μm would be sufficient for their communication. The graded rescue of each neuromodulator by expression in specific neurons or groups of neurons suggests that both the quantity and the source of neuromodulators contribute to their function (Flavell, 2013).

The circuits that drive short roaming and dwelling states in the absence of serotonin and PDF remain to be defined, as do the neurons required for the individual motor patterns of roaming and dwelling. Importantly, each neuron in the roaming and dwelling circuit has other behavioral functions, which will provide challenges for mapping the circuits. For example, the PDF-producing AVB neuron is essential for coordinated forward locomotion, which it controls through chemical and electrical synapses onto motor neurons. Thus, AVB is required for coordinated forward movement at rapid timescales and releases PDF-1 peptides to regulate locomotion over longer durations. Because neurons like AVB are multifunctional, precise manipulation of neuronal signaling molecules may be needed to disentangle the activities of neurons, synapses, and modulators on behavior (Flavell, 2013).

Multifunctionality is also a property of the PDF and serotonin neuromodulators, which each affect a variety of behaviors by mobilizing different sets of neurons. For example, in males, PDF-1 produced by AIM neurons signals to the PDFR-1-expressing neurons URY, PQR, and PHA to stimulate mate search. Although these neurons are all present in the hermaphrodite, they appear unimportant in roaming and dwelling behavior. Similarly, serotonin regulates feeding and egg laying using receptors and neurons distinct from those defined in this study. In summary, neither neurons, neuromodulators, nor behaviors are subsets of one another-each represents a separate functional organization within the nervous system (Flavell, 2013).

Dwelling and roaming are persistent behaviors that last for several minutes. The endogenous calcium signals in serotonergic NSM neurons are long lasting, but not identical to the behavioral states-NSM calcium transients last about 1 min but predict dwelling states for several minutes thereafter. Either optogenetic excitation of NSM or optogenetic inhibition of its MOD-1-expressing target neurons with ARCH was sufficient for persistent dwelling states. As ARCH should have a direct hyperpolarizing effect on the MOD-1-expressing neurons, a persistent circuit state for dwelling may be induced by transient neuronal inhibition. The failure of mod-1::ARCH to induce long-lasting dwelling states in mod-1 and tph-1 mutants suggests that continued serotonergic signaling maintains dwelling states (Flavell, 2013).

The origin of the endogenous NSM calcium signals is unknown. NSM's position in the pharynx suggests that it could detect cues associated with feeding; in addition, NSM calcium levels are indirectly regulated by attractive and repulsive odors and the biogenic amine tyramine. Thus, NSM could detect both nutrients and sensory cues relevant to roaming and dwelling. However, nutrients and sensory cues also regulate ASI and AIY, so there are many neurons in the serotonin and PDF circuits that provide possible entry points for regulation. Understanding the relationship between egl-4, which functions in sensory neurons to promote dwelling, and the circuit described in this study might clarify how nutrient and sensory cues are coupled to behavioral transitions (Flavell, 2013).

The C. elegans PDF receptor is coupled to G&alpah;s and cAMP production, so for optogenetic imitation of PDFR-1 activation, this study employed the bacterial light-activated adenylyl cyclase BlaC. BlaC activation in PDFR-1-expressing neurons triggered roaming states that lasted for several minutes after light cessation should terminate its catalytic activity. Persistent roaming could result from sustained cAMP levels, sustained activation of the cAMP-dependent protein kinase PKA, or sustained phosphorylation of PKA targets. Taking a broader view, the slow time course of neuromodulatory G protein signaling is well suited to convert short-lasting electrical signals into longer-lasting biochemical and behavioral states (Flavell, 2013).

A role for PDF signaling in promoting roaming, an arousal state in C. elegans, is reminiscent of the ability of PDF to promote arousal during waking states in Drosophila. In flies, PDF-expressing neurons integrate the circadian cycle, light levels, and modulatory octopamine and dopamine inputs to regulate PDF release and arousal. A mammalian neuropeptide, vasoactive intestinal peptide (VIP), has a similar role in stimulating arousal downstream of light inputs and neuromodulation in the suprachiasmatic nucleus. PDF receptors and VIP receptors are similar in sequence, suggesting that these neuropeptide systems may have similar or even conserved roles in arousal states (Flavell, 2013).

More generally, the circuit logic of roaming and dwelling resembles the logic of hypothalamic and brainstem circuits that control discrete mammalian sleep and wake behaviors. The transitions between wake, REM, and non-REM sleep are controlled by neuropeptides and biogenic amines produced in hypothalamic and brainstem nuclei. As is seen in roaming and dwelling, each state inhibits the others in a switch-like fashion, and loss of the neuromodulators leads to destabilized and truncated behavioral states. It is suggested that these features may be signatures of a variety of discrete behavioral states (Flavell, 2013).

Insect Pigment-dispersing factor

The pigment-dispersing hormone (PDH) family of neuropeptides comprises a series of closely related octadecapeptides, isolated from different species of crustaceans and insects. It can be demonstrated immunocytochemically in neurons in the central nervous system and optic lobes of some representatives of these groups. In the blowfly Phormia terraenovae, tissue extracts were tested in a bioassay: extracts of blowfly brains exhibit PDH-like biological activity, causing melanophore pigment dispersion in destalked (eyestalkless) specimens of the fiddler crab Uca pugilator. Using standard immunocytochemical techniques, a small number of pigment-dispersing hormone-immunoreactive (PDH-IR) neurons innervating optic lobe neuropil in the blowfly and the cockroach Leucophaea maderae could be demonstrated. In the blowfly the cell bodies of these neurons are located at the anterior base of the medulla. At least eight PDH-IR cell bodies of two size classes can be distinguished: 4 larger and 4 smaller. Branching immunoreactive fibers invade three layers in the medulla neuropil, and one stratum distal and one proximal to the lamina synaptic layer. A few fibers can also be seen invading the basal lobula and the lobula plate. The fibers distal to the lamina appear to be derived from two of the large PDH-IR cell bodies, which also send processes into the medulla. These neurons share many features in their lamina-medulla morphology with the serotonin immunoreactive neurons LBO-5HT described earlier. It could be demonstrated by immunocytochemical double labeling that the serotonin and PDH immunoreactivities are located in two separate sets of neurons. In the cockroach optic lobe PDH-IR processes were found to invade the lamina synaptic region and form a diffuse distribution in the medulla. The numerous cell bodies of the lamina-medulla cells in the cockroach are located basal to the lamina in two clusters. Additional PDH-IR cell bodies can be found at the anterior base of the medulla. The distribution and morphology of serotonin-immunoreactive neurons in the cockroach lamina have been found to be very similar to the PDH-IR neurons. Hence, it is tempting to speculate that in both species the PDH- and serotonin-immunoreactive neurons are functionally coupled with common follower neurons. These neurons may be candidates for regulating large numbers of units in the visual system (Nassel, 1991).

Pigment-dispersing hormone immunoreactive (PDHIR) neurons could be detected in the brain and ventral ganglia of the blowfly Phormia terraenovae. PDHIR neurons are found in the optic lobe. Their processes supply the lamina, medulla and lobula complex bilaterally. Large PDHIR cell bodies in the protocerebrum have processes in the proto- and tritocerebrum and axons to the aorta wall and foregut. Eight pairs of PDHIR neurons are found dorsally and three pairs ventrally in the fused abdominal neuromeres; one pair is located ventrally in each of the thoracic neuromeres. The ventral abdominal PDHIR neurons are efferents that innervate the hindgut. PDHIR neurons may play different functional roles as neurohormones or neuromodulators in different parts of the nervous system and its peripheral targets (Shiga, 1993).

An antiserum against the crustacean neuropeptide pigment-dispersing hormone stains a small set of neurons in the optic lobes of several hemimetabolous and holometabolous insects. These cells, the primary branches of which in the optic lobe lie in the accessory medulla, fulfill several criteria predicted for neurons of the circadian clock. For example, in fruit flies they express timeless and period, which are two molecular components of the circadian pacemaker. To test whether pigment-dispersing hormone fulfills a circadian function in the cockroach Leucophaea maderae, 150 fmol of synthetic peptide was injected into the vicinity of the accessory medulla. This results in a stable phase-dependent resetting of the phase of the circadian locomotor activity rhythm, which depends on the amount of pigment-dispersing hormone injected. The resulting phase-response curve differs from that obtained with light pulses, suggesting that pigment-dispersing hormone-immunoreactive neurons are not part of the visual input pathway to the pacemaker but an integral part of it and/or part of a nonphotic input into the clock (Petri, 1997).

The accessory medulla with its associated pigment-dispersing hormone-immunoreactive neurons appears to be the pacemaker that controls the circadian locomotor activity rhythm of the cockroach Leucophaea maderae. To permit studies at the level of individual, identified, pacemaker neurons, specific long-term primary cell cultures of fully differentiated adult neurons of the accessory medulla were developed. As judged from soma diameter distribution, the cultures contain an unbiased representation of apparently all neuronal types of the accessory medulla. The cultured cells survive and grow processes for more than 2 months, with or without additional hemocyte coculturing. However, a strong positive effect on initial outgrowth was observed with hemocyte coculturing. At least six different morphological cell types of the accessory medulla could be distinguished in vitro. Among these, only one cell type, the monopolar type C cell, was recognized in vitro with an antiserum against the neuropeptide pigment-dispersing hormone. Thus, the identifiable monopolar type C cells are candidates for circadian pacemaker neurons (Petri, 1999)

Pigment-dispersing factor-immunoreactive circadian pacemaker cells, which arborize in the accessory medulla, control circadian locomotor activity rhythms in Drosophila as well as in the cockroach Leucophaea maderae via unknown mechanisms. Circadian pacemaker candidates of the accessory medulla of the cockroach produce regular interspike intervals. Therefore, the membrane potential of the cells oscillates with ultradian periods. Most or all oscillating cells within the accessory medulla are coupled via synaptic and nonsynaptic mechanisms, forming different assemblies. The cells within an assembly share the same ultradian period (interspike interval) and the same phase (timing of spikes), whereas cells between assemblies differ in phase. Apparently, the majority of these assemblies are formed by inhibitory GABAergic synaptic interactions. Application of pigment-dispersing factor phase locked and thereby synchronized different assemblies. The data suggest that pigment-dispersing factor inhibits GABAergic interneurons, resulting in disinhibition and phase locking of their postsynaptic cells, which previously belonged to different assemblies. These data suggest that phase control of action potential oscillations in the ultradian range is a main task of the circadian pacemaker network. It is hypothesized that neuropeptide-dependent phase control is used to gate circadian outputs to locomotor control centers (Schneider, 2005).

Crustacean Pigment-dispersing hormone

The pigment-dispersing hormone (PDH) is produced in the eyestalks of Crustacea where it induces light-adapting movements of pigment in the compound eye and regulates the pigment dispersion in the chromatophores. To study this hormone at the mRNA level, cDNA encoding PDH in the crayfish Orconectes limosus was cloned and sequenced. The structure of the PDH preprohormone consists of a signal peptide, a PDH precursor-related peptide (PPRP) and the highly conserved PDH peptide at the carboxy-terminal end. In situ hybridization in combination with immunocytochemistry reveal four cell clusters expressing PDH in the optic ganglia of the eyestalk. Three clusters stain both with the PDH cRNA probe and the PDH antiserum, however, the perikarya in the lamina ganglionaris (LG) only stain with the PDH antiserum, suggesting the presence of a PDH-like peptide in the LG (de Kleijn, 1993).

A cDNA library was established from the eyestalk ganglia of the blue crab Callinectes sapidus. Screening resulted in the isolation of a clone [497 bp excluding poly(A) tail] that encodes a beta-PDH previously found in several crustacean species. It displays high sequence similarity with a clone isolated from an eyestalk cDNA library of the shore crab Carcinus maenas, indicating the close phylogenetic relationship of both species. A second clone [414 bp exclusive of the poly(A) tail] encodes a novel beta-PDH analog that displays 400-fold less potency in crab bioassays. Both cDNAs encode open reading frames of 234 bp for the prepropeptides, consisting of signal peptides, PDH-precursor-related peptides, and PDH sequences (Klein, 1994).

Neurons immunoreactive with antisera against the crustacean peptide beta-pigment dispersing hormone fulfill several anatomical criteria proposed for circadian pacemakers in the brain of the cockroach Leucophaea maderae. These include position of somata, projections to the lamina and midbrain and possible coupling pathways between the two pacemakers through commissural fibers. In behavioral experiments combined with lesion studies and immunocytochemical investigations an examination was carried out to see whether the presence of pigment-dispersing hormone-immunoreactive arborizations in the midbrain of the cockroach correlate with the presence of circadian locomotor activity. No rhythm was detected in any animal for at least 12 days after severing both optic stalks. Within the same time period, pigment-dispersing hormone-immunoreactive fibers in the midbrain disappeared. Two to seven weeks after the operation some of the cockroaches regained circadian locomotor activity, while others remained arrhythmic. In all cockroaches that regained rhythmic behavior, pigment-dispersing hormone-immunoreactive fibers had regenerated and had largely found their original targets within the brain. In all arrhythmic cockroaches, either none or very little regeneration had occurred. The period of the regained circadian activity inversely correlates with the number of regenerated immunoreactive commissural fibers. These data provide further evidence for the involvement of pigment-dispersing hormone-immunoreactive neurons in circadian clocks of orthopteroid insects (Stengl, 1994).

The cDNAs encoding the precursors of a chromactive crustacean hormone, Pigment Dispersing Hormone (PDH) of the shrimp Penaeus vannamei, were studied by PCR and molecular cloning. Three different cDNAs were isolated and sequenced. The PDH precursor consists of a putative 22- or 23-amino acid signal peptide, a 34-amino acid PDH-Precursor Related Peptide (PPRP) of unknown function, and the 18-amino acid mature PDH. The deduced mature PDH amino acid sequences are identical except the change of a Leucine by an Isoleucine in one variant and are very similar to those of other species. The signal peptides appear highly variable. The variability between the PPRP sequences is low between the different species, suggesting that this peptide may have a physiological role (Desmoucelles-Carette, 1996).

Three chromatophorotropic neuropeptide hormones were purified from an aqueous extract of the sinus glands of the kuruma prawn Penaeus japonicus by two steps of reverse-phase HPLC and their amino acid sequences were determined. One of them has been found to show pigment concentrating activity and to have an amino acid sequence identical to that of the known red pigment concentrating hormone (RPCH); therefore, it was named Pej-RPCH. The other two peptides show pigment dispersing hormone (PDH) activity and have been named Pej-PDH-I and -II. They both consist of 18 amino acid residues with a free amino-terminus and an amidated carboxyl-terminus, the sequences of Pej-PDH-I and -II being NSELINSLLGIPKVMTDAamide and NSELINSLLGLPKFMIDAamide, respectively. Three amino acid residues at positions 11, 14, and 16 differ between the two PDHs. Pej-PDH-II is about 5-, 7-, and 10-fold more potent than Pej-PDH-I for erythrophores, xanthophores, and melanophores, respectively. The major reason for the difference in potency between the two PDHs can be attributed to differences in residues at position 16. Additionally, each is produced by single individuals. The order of sensitivity of the four types of chromatophores to Pej-RPCH and both PDHs has been found to be erythrophores = xanthophores, which are greater than melanophores, which, in turn, are greater than leukophores (Yang, 1999).

Molluscan Pigment-dispersing hormone

By using an antiserum raised against a crustacean beta-pigment-dispersing hormone (PDH), the distribution and chemical neuroanatomy of PDH-like immunoreactiveneurons has been investigated in the central nervous system of the gastropod snails, Helix pomatia and Lymnaea stagnalis. The number of immunoreactive cells in the Helix central nervous system is large (700-900), whereas in Lymnaea, only a limited number (50-60) of neurons show immunoreactivity. The immunostained neurons in Helix are characterized by rich arborizations in all central ganglia and reveal massive innervation of all peripheral nerves and the neural (connective tissue) sheath around the ganglia and peripheral nerve trunks. A small number of Helix nerve cell bodies in the viscero-parietal ganglion complex are also found to be innervated by PDH-like immunoreactive processes. Hence, a complex central and peripheral regulatory role, including neurohormonal actions, issuggested for a PDH-like substance in Helix, whereas the sites of action may be more limited in Lymnaea (Elikes, 1999).

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).

Genetic dissection of stress-induced reproductive arrest in Drosophila melanogaster females

By genetic manipulations, the roles played by insulin-producing cells (IPCs) in the brain and their target, the corpora allata (CA), was studied for reproductive dormancy in female Drosophila melanogaster that was induced by exposing them to a combination of low temperature (11 ° C), short-day photoperiod (10L:14D) and starvation (water only) for 7 days immediately after eclosion (dormancy-inducing conditions). Artificial inactivation of IPCs promotes, whereas artificial activation impedes, the induction of reproductive dormancy. A transcriptional reporter assay reveals that the IPC activity is reduced when the female flies are exposed to dormancy-inducing conditions. The photoperiod sensitivity of reproductive dormancy is lost in pigment-dispersing factor (pdf), but not cry, mutants, suggesting that light input to IPCs is mediated by pdf-expressing neurons. Genetic manipulations to upregulate and downregulate insulin signaling in the CA, a pair of endocrine organs that synthesize the juvenile hormone (JH), decrease and increase the incidence of reproductive dormancy, respectively. These results demonstrate that the IPC-CA axis constitutes a key regulatory pathway for reproductive dormancy (Ojima, 2018).

REFERENCES

Search PubMed for articles about Drosophila Pigment-dispersing factor

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date revised: 1 January 2024

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