Drosophila NIH Public Access from PDF Neurons Determines ... · (pdf > 2x t-PDF, pdf > 4x t-PDF or pdf > 6x t-PDF) for examination of dose-dependent effects of PDFR activation. Flies

Autoreceptor Modulation of Peptide/Neurotransmitter Co-releasefrom PDF Neurons Determines Allocation of Circadian Activity inDrosophila

Charles Choi1, Guan Cao1, Anne K. Tanenhaus4, Ellena v. McCarthy1, Misun Jung1, WilliamSchleyer1, Yuhua Shang5, Michael Rosbash5, Jerry C.P. Yin4, and Michael N. Nitabach1,2,3,*

1Department of Cellular and Molecular Physiology, Yale School of Medicine, 333 Cedar Street,New Haven, CT 06520, USA2Department of Genetics, Yale School of Medicine, 333 Cedar Street, New Haven, CT 06520,USA3Program in Cellular Neuroscience, Neurodegeneraton and Repair, Yale School of Medicine, 333Cedar Street, New Haven, CT 06520, USA4Departments of Genetics and Neurology, University of Wisconsin, Madison, 3434 Genetics/Biotech, 425 Henry Mall, Madison, WI 52706, USA5Howard Hughes Medical Institute and National Center for Behavioral Genomics, Department ofBiology, Brandeis University, Waltham, MA 02454, USA

AbstractDrosophila melanogaster flies concentrate behavioral activity around dawn and dusk. Thisorganization of daily activity is controlled by central circadian clock neurons, including the lateralventral pacemaker neurons (LNvs) 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. While LNvs also expressfunctional PDFR, the role of these autoreceptors has remained enigmatic. Here we show that (1)PDFR activation in LNvs shifts the balance of circadian activity from evening to morning, similarto behavioral responses to summer-like environmental conditions and (2) this shift is mediated bystimulation of the Ga,s-cAMP pathway and a consequent change in PDF/neurotransmitter co-release from the LNvs. These results suggest a novel mechanism for environmental control of theallocation of circadian activity and provide new general insight into the role of neuropeptideautoreceptors in behavioral control circuits.

IntroductionDrosophila melanogaster flies concentrate their behavioral activity around dawn and dusk(Helfrich-Forster, 2000) and sleep mostly at night and in the middle of the day (Hendricks etal., 2000; Shaw et al., 2000). In 12hr:12hr light:dark (LD) conditions, their daily morningand evening activity bouts begin before the environmental transitions, and the the timing and

© 2012 Elsevier Inc. All rights reserved.*Corresponding Author: [email protected].

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NIH Public AccessAuthor ManuscriptCell Rep. Author manuscript; available in PMC 2012 September 04.

Published in final edited form as:Cell Rep. 2012 August 30; 2(2): 332–344. doi:10.1016/j.celrep.2012.06.021.

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amplitude of these activity bouts are influenced by daily dynamics of ambient temperatureand light (Majercak et al., 1999; Vanin et al., 2012; Zhang et al., 2010b). These morning andevening activity bouts persist in constant darkness (DD), indicating the sufficiency of theinternal timekeeping system for their generation. Both entrained activity rhythms in LD andfree-running rhythms in DD are driven by a network of central circadian clock neurons(Nitabach and Taghert, 2008).

Of the approximately 75 bilateral pairs of circadian neurons, only small and large lateralventral circadian clock neurons (sLNvs and lLNvs; 9 bilateral pairs) secrete the neuropeptidePDF (Pigment Dispersing Factor) (Helfrich-Forster, 1995; Renn et al., 1999). lLNvs projectto the optic lobes and the accessory medulla (AMe) and sLNvs project to the doral brainregion (Helfrich-Forster et al., 2007), where PDF secretion is likely circadian, reaching amaximum at about dawn (Cao and Nitabach, 2008; Park et al., 2000). Secreted PDF actingon clock cells expressing the PDF receptor (PDFR) is required for normal circadian function(Hyun et al., 2005; Im and Taghert, 2010; Lear et al., 2005; Lear et al., 2009; Mertens et al.,2005; Zhang et al., 2010a). PDFR is expressed more broadly than PDF in the circadiannetwork: the receptor is expressed in the PDF-negative dorsal clock neurons (dorsal neurongroups (DNs) and dorsolateral neuron group (LNds)) as well as in the PDF-positive sLNvsand in some PDF-positive lLNvs (Im and Taghert, 2010; Kula-Eversole et al., 2010; Shaferet al., 2008). PDFR is a G protein-coupled receptor (Hyun et al., 2005; Lear et al., 2005;Mertens et al., 2007), which couples to adenylyl cyclase/cAMP in HEK293 cells and allclock neurons expressing PDFR but the lLNvs (Shafer et al., 2008). PDFR maintains someintracelluar signaling specificity by coupling to specific adenylyl cyclase isoforms that differin different clock cell groups (Duvall and Taghert, 2012). PDF signaling is critical fornormal circadian behavior, as genetic ablation of PDF or PDFR leads to loss of circadianmorning activity and phase-advanced evening activity in LD as well as weakened free-running rhythms with short periods in DD (Hyun et al., 2005; Lear et al., 2005; Mertens etal., 2005; Renn et al., 1999). Interestingly, the circadian functions of PDF and PDFR aresimilar to those of VIP and VIPR (Vasoactive Intestinal Peptide and VIP Receptor), whichare expressed by clock neurons in the mammalian suprachiasmatic nucleus (SCN) and arerequired in mice for robust circadian wheel-running rhythms and synchronized rhythms ofclock gene expression (Aton et al., 2005; Colwell et al., 2003; Harmar et al., 2002;Maywood et al., 2006).

Recent studies indicate that PDF secretion from sLNvs to the PDF-negative dorsal clockclock neurons is critical for generating circadian morning activity and robust free-runninglocomotor activity rhythms (Grima et al., 2004; Lear et al., 2009; Shafer and Taghert, 2009;Stoleru et al., 2005; Zhang et al., 2010a). The phase of circadian morning activity iscorrelated with the phase of daily rhythmic PDF secretion (Wu et al., 2008b), suggestingthat PDF rhythms encode timing information for circadian morning activity. In addition, themagnitude of PDF signaling to dorsal clock neurons can also influence the pace of free-running locomotor rhythms (Choi et al., 2009; Wulbeck et al., 2008). These studies indicatea key role of PDF signaling from LNvs to PDF-negative dorsal clock neurons in organizingdaily activity.

What remains enigmatic is the function of PDFR autoreceptors in PDF-positive LNv clockneurons. It has been suggested that these autoreceptors contribute to stronger free-runningrhythms in DD, as PDFR null-mutant flies with rescue of PDFR in all clock neurons exhibitmore robust free-running rhythms than flies with PDFR rescue solely in PDF-negative clockneurons (Lear et al., 2009). Furthermore, there is evidence that lLNvs mediate light-inducedphase-shifts in late night, and it has been suggested that PDF signals from lLNvs to sLNvsunderlie these phase-shifts (Shang et al., 2008). However, direct evidence for a specific roleof LNv PDFR autoreceptor activation has remained absent. Here, we show that sLNv PDFR

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activation determines the allocation of daily circadian activity between the morning andevening by engaging the Gα,s-cAMP pathway, depolarizing the sLNvs, and modulatingsecretion of PDF and classical synaptic neurotransmitter. These results not only resolve thequestion of a functional role for PDF autoreceptors in the control of circadian acitivityrhythms, but also provide general insight into neuropeptide autoreceptor modulation ofbehavioral control circuits.

ResultsPDFR Activation In LNv Pacemaker Cells Increases “Morningness” of Daily Activity

In order to assess the role of PDFR autoreceptors in the PDF-positive LNv pacemakerneurons in organizing daily circadian activity, we cell-autonomously activated LNv PDFRby transgenic expression of membrane-tethered PDF (t-PDF) (Choi et al., 2009). t-PDF is agenetically encoded PDFR agonist with active PDF covalently linked to the cell surface viaa C terminal GPI (glycophosphatidylinositol) anchor whose lipid chains are intercalated inthe outer leaflet of the plasma membrane (Choi et al., 2009). GPI-anchoring limits the actionof t-PDF to the cell in which it is expressed, where it cell-autonomously activates nativePDFR without affecting PDFR on neighboring cells (Choi et al., 2009). Using the GAL4-UAS expression system, t-PDF expression can be genetically targeted to specificallyactivate PDFR in selected cells in vivo (Choi et al., 2009). We combined the LNv-specificpdf-GAL4 driver (Renn et al., 1999) with UAS-t-PDF effector transgene to induce t-PDFexpression specifically in the PDF-positive LNvs. Two, four or six independentchromosomal insertions of UAS-t-PDF transgene were combined with the pdf-GAL4 driver(pdf > 2x t-PDF, pdf > 4x t-PDF or pdf > 6x t-PDF) for examination of dose-dependenteffects of PDFR activation. Flies with pdf-GAL4 and six copies of UAS-t-SCR, an inertisoform of UAS-t-PDF with the amino acid sequence of the PDF peptide moiety scrambled(Choi et al., 2009), were used as negative controls (pdf > 6x t-SCR).

In 12 hr/12hr light/dark (LD) conditions, LNv PDFR activation leads to increased morninganticipatory activity before lights-on, with greater t-PDF expression resulting incorrespondingly greater increased morning anticipatory activity (Figure 1A and 1D). Incontrast, the locomotor profile during the light phase is little affected (Figure 1A). Activityprofiles during the first day after release into constant darkness (DD1) reveal free-runningcircadian rhythmic activity (Lear et al., 2009). Like in LD, flies in DD exhibit a dose-dependent effect of LNv PDFR activation, with greater t-PDF expression increasing the ratioof circadian activity in the subjective morning to that in the subjective evening (Figure 1B,1F), even over multiple days in DD (Figure 1C). This increase in “morningness” ofcircadian activity is primarily due to increased morning activity in this experiment, but insome cases, it is mediated by a decrease in evening activity (see, e.g., Figure 2A, third panel,second row, Supplemental Table 1). LNv PDFR activation accelerates free-running rhythmsin DD (Figure 1C, 1F), which may contribute to the increased morning anticipatory activityas a consequence of phase advance (Figure 1A, 1D). These results demonstrate thatincreased PDFR activation in LNvs has two effects: shifting the allocation of daily activityin favor of morning and advancing the phase of morning activity.

PDFR-mediated Increased Morningness Requres Gα,s-cAMP SignalingPDFR activation increases intracellular cAMP in HEK293 cells and most clock neurons(including sLNvs), indicating that PDFR is coupled to Gα,s (Choi et al., 2009; Mertens etal., 2005; Shafer et al., 2008). In order to assess whether the increased morningness inducedby LNv PDFR activation is mediated by Gα,s-cAMP, we co-expressed t-PDF with a numberof different transgenes that modulate this pathway. Gα,s-11 is an eleven residue peptide thatcompetitively inhibits receptor-Gα,s interaction (Yao and Carlson, 2010) and PDE8 is a

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cAMP-specific phosphodiesterase that reduces intracellular cAMP levels whenoverexpressed (Day et al., 2005). Co-expression of either of these transgenes inhibits thebehavioral effects of t-PDF expression (Figure 2A). The t-PDF effect was inhibited also byRNAi knockdown of Gα,s (Figure 2B, Supplemental Figure 1). Gα,s[GTP] contains a pointmutant that impairs GTPase activity and renders the G protein constitutively active(Connolly et al., 1996; Wolfgang et al., 1996). Expression of either 4x t-PDF or Gα,s[GTP]increases morningness, and there was no additive effect of co-expression of these transgenes(Figure 2C). These genetic interactions between t-PDF and the loss-of-function or gain-of-function Gα,s-cAMP pathway perturbations indicate that LNv PDFR activation engages theGα,s-cAMP pathway to modulate circadian morningness. Because lLNv PDFR is notcoupled to adenylyl cyclase/cAMP (Shafer et al., 2008), these results support theinterpretation that increased morningness is mediated by PDFR activation in sLNvs and notlLNvs.

DH31R Activation In LNvs Does Not Increase MorningnessIn addition to PDFR, LNvs express the receptor for the fly homologue of CGRP, DH31(Shafer et al., 2008). DH31R (CG17415), like PDFR, is a class B1 neuropeptide receptorcoupled to the Gα,s-cAMP pathway (Johnson et al., 2005), and LNvs respond to bathapplication of DH31 with increased intracellular cAMP (Shafer et al., 2008). In order toassess whether DH31R activation in LNvs also increases morningness, we expressed t-DH31with two different insertion combinations of 6 copies of UAS-t-DH31 (pdf > 6x t-DH31).We have previously shown that t-DH31 strongly activates DH31R in cell culture (Choi etal., 2009). Unlike PDFR activation of LNvs, DH31R activation of LNvs increases neithermorning anticipatory activity in LD nor morningness in DD and rather decreasesmorningness (Figure 3A, 3B, 3E). DH31R activation of LNvs does accelerate free-runningrhythms but substantially less than PDFR activation (Figure 3C, 3F). This indicates thatLNvs can distinguish activation of PDFR from DH31R, and that increased morningness isspecific for activation of PDFR. This specificity could be in part due to the differentsensitivity of LNvs to PDF and DH31, as revealed by studies showing cAMP increase tobath-applied DH31 in sLNvs and lLNvs, but cAMP increases to PDF only in sLNvs (Shaferet al., 2008). In addition, it has recently been shown that the adenylyl cyclase isoform AC3and the AKAP (A-kinas anchoring protein) cAMP signaling pathway scaffolding proteinnervy are both required for cAMP increases of sLNvs to bath-applied PDF but not to DH31(Duvall and Taghert, 2012). This suggests that PDFR activation in LNvs modulates the dailyallocation of circadian activity by intracellular signal transduction pathways that aresegregated from those engaged by DH31R. Our in vivo results are consistent with these invitro studies, and indicate that daily allocation of activity is specifically regulated by thePDF/PDFR signal transduction pathway and that LNvs can functionally distinguish betweenactivation of the related PDFR and DH31R class B1 receptors.

PDFR Activation Cell-Autonomously Depolarizes sLNvsPrevious studies show that LNv resting membrane potential (RMP) cycles over the course ofthe day with greatest depolarization around dawn (Cao and Nitabach, 2008; Sheeba et al.,2008b), and that altering the electrophysiological properties of LNvs leads to modifiedcircadian locomotor rhythms (Nitabach et al., 2002; Nitabach et al., 2006; Sheeba et al.,2008a; Wu et al., 2008a; Wu et al., 2008b). These studies indicate that the electricalproperties of the LNvs are integral to their role in circadian timekeeping and output. Also,the two LNv subsets play distinct roles in generating circadian rhythms and arousal, asshown in both prior studies and the results described above (Cusumano et al., 2009; Pariskyet al., 2008; Shafer and Taghert, 2009; Shang et al., 2008; Sheeba et al., 2008a; Stoleru etal., 2005). Accordingly, we asked whether PDFR activation alters either lLNv or sLNvmembrane activity around dawn and thereby modifies cellular output to increase

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morningness. To address this question, we performed whole-cell patch-clamp of the cellbodies of sLNvs and lLNvs, in pdf > 6x t-PDF and pdf > 6x t-SCR flies at ZT22–23, beforedawn and around the time morning activity begins.

sLNvs of pdf > 6x t-SCR flies display regular ~1 Hz membrane potential oscillations drivenby synchronous rhythmic synaptic inputs and exhibit average RMP of −55.9±4.57 mV(Figure 4A, 4B). lLNvs of these flies exhibit relatively less regular RMP oscillations at thiscircadian time with average RMP of −44.5±1.10 mV (Figure 4A, 4B). These observations oft-SCR-expressing control flies are consistent with eletrophysiological measurements ofwild-type LNvs at this circadian time (Cao and Nitabach, 2008). There are no differences inthe average RMP or the strength of membrane potential oscillations between the lLNvs ofpdf > 6x t-PDF and pdf > 6x t-SCR flies (Figure 4A, 4B). However, the sLNvs of pdf > 6x t-PDF flies are significantly more depolarized than the sLNvs of pdf > 6x t-SCR flies (Figure4A, 4B). To determine whether these changes in sLNv membrane properites are a cell-autonomous consequence of PDFR activation, we repeated these measurements in thepresence of bath TTX, which blocks action potential-dependent network activity. Even inthe absence of network activity, pdf > 6x t-PDF sLNvs were more depolarized than pdf > 6xt-SCR sLNvs, indicating that PDFR activation depolarizes sLNvs by altering their intrinsicmembrane properties (Figure 4D, 4E). This depolarization of sLNvs in pdf > 6x t-PDF fliesmay in part reflect phase-advanced circadian RMP (and hence, PDF secretion) rhythms,since resting membrane potential of sLNvs progressively depolarizes from ZT16 to ZT0(Cao and Nitabach, 2008), consistent with the behavioral phase advance in these flies(Figure 1C, 1F). However, it may also reflect a more depolarized sLNv potential and greaterpeak neural output, independent of phase. The depolarization of sLNvs induced by PDFRactivation is an interesting parallel to the depolarization of mammalian SCN neuronsinduced by VIPR activation (Pakhotin et al., 2006).

In addition to becoming depolarized, regular membrane potential oscillations seen in thesLNvs of pdf > 6x t-SCR flies are absent in the sLNvs of pdf > 6x t-PDF flies, as quantifiedby autocorrelation analysis (Figure 4A, 4C). These network dependent oscillations are alikely consequence of rhythmic synaptic activation of nicotinic acetylcholine receptors(nAChRs), as has been established for lLNvs (McCarthy et al., 2011). Accordingly,depolarization towards the reversal potential for nAChR-mediated synaptic currents ispredicted to decrease the magnitude of oscillation, consistent with our observations (Figure4D). Cell-autonomous and sLNv-specific depolarization and suppression of membranepotential oscillation are PDFR-driven changes in sLNv cellular physiology that alter sLNvneural output to downstream targets controlling daily allocation of circadian activity.

PDF Output From LNvs Is Required For PDFR-Induced MorningnessWhat potential sLNv output pathways could be altered by PDFR-induced depolarization?PDF secretion by sLNvs is necessary for circadian morning activity (Grima et al., 2004;Renn et al., 1999; Shafer and Taghert, 2009; Stoleru et al., 2004; Stoleru et al., 2005). PDFRexpression in only a restricted subset of dorsal clock neurons (DNs) is sufficient forcircadian morning activity, while PDFR expression only in LNvs is not (Lear et al., 2009;Zhang et al., 2010a). This indicates that PDF signals from sLNvs to DNs are sufficient forcircadian morning activity and suggest a possible modulatory role of PDF secretion bysLNvs to DNs for controlling morningness. To test this, we activated PDFR in LNvs usingtwo or four copies of UAS-t-PDF in pdf01 null-mutants. This did not lead to any rescue ofmorning anticipation or robust circadian activity peaks (Figure 5A), indicating that PDFsecretion by LNvs is a requirement for increased morningness induced by LNv PDFRactivation. However, t-PDF expression and consequent cell-autonomous PDFR activationsolely in PDF-negative clock neurons of pdf01 null-mutant flies rescues circadian morningactivity (Figure 5B). This is consistent with the previous observation that PER rescue in per0

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null-mutant flies solely in a subset of DNs rescues morning anticipation in LD, where thesource of their PDF input is the clock-less LNvs (Zhang et al., 2010b). This rescue ofcircadian morning activity thus indicates that PDFR activation in PDF-negative clockneurons without LNv PDFR activation or LNv PDF secretion is sufficient for robustcircadian morning activity. Furthermore, when we assessed PDF accumulation byimmunocytochemistry in the sLNv dorsal terminals at dawn, we found higher levels of PDFaccumulation in the sLNv dorsal terminals of pdf > 6x t-PDF flies than pdf > 6x t-PDF flies(Figure 5C). Since pdf > 6x t-PDF flies are more depolarized at this time (Figure 4B), it islikely that this greater PDF accumulation accompanies increased sLNv PDF output (see (Wuet al., 2008b)). Taken together, these results reinforce the idea that endogenous PDFsignaling to DNs is necessary for the generation of circadian morning activity (Lear et al.,2009; Zhang et al., 2010a), and suggest that increased PDF output by sLNvs is involved inthe PDFR autoreceptor-induced increase in morningness.

Classical Neurotransmitter Output From LNvs Is Required For PDFR-Induced MorningnessThere is some evidence that classical neurotransmitters may also participate with PDF inLNv circadian output, although whether such signals are instructive to the daily allocation ofactivity remains unclear. Small clear vesicles (SCVs) that house classical neurotransmittersare co-localized with PDF-containing dense-core vesicles (DCVs) in the sLNv dorsalprojections (Miskiewicz et al., 2004; Yasuyama and Meinertzhagen, 2010). Functionally,electrical hyperexcitation of LNvs enhances the robustness of behavioral rhythms in pdf01

null-mutants (Sheeba et al., 2008c), and blocking LNv classical neurotransmitters withtetanus toxin (TnTLC) partially rescues cellular and behavioral rhythms in constant lightconditions (Umezaki et al., 2011), although it has no effect on free-running rhythms inconstant darkness (Kaneko et al., 2000). To test whether classical non-peptide synapticneurotransmitter secretion by LNvs also plays a role in PDFR-induced morningness, weused a 10x UAS-TnTLC transgene encoding tetanus toxin light chain with 10x UASupstream promoter for improved expression (10x UAS-TnTLC, gift from Brian McCabe).TnTLC cleaves neuronal Synaptobrevin, a SNARE protein necessary for calcium-dependentclassical neurotransmitter release (Sweeney et al., 1995). We co-expressed t-SCR or t-PDFwith TnTLC in LNvs to block LNv synaptic output to determine whether LNv PDFRactivation requires classical neurotransmission to increase morningness.

pdf > 4x t-SCR + TnTLC flies maintain robust daily circadian activity peaks and free-running rhythms (Figure 6), unlike pdf01 null-mutant flies (see controls in Figure 5 forexample). This confirms the specificity of TnTLC for classical neurotransmitter secretionand lack of interference with PDF secretion. However, LNv TnTLC expression almostcompletely suppresses increased morningness induced by activation of PDFR in LNvs.While pdf > 4x t-PDF flies exhibit increased morning anticipatory activity and morningnesscompared to pdf > 4x t-SCR, identical to our earlier experiments (Figure 1), pdf > 4x t-PDF+ TnTLC flies exhibit dramatically less morning anticipatory activity than pdf > 4x t-PDFflies (Figure 6A, 6D) as well as completely suppressed increase in morningness (Figure 6B,6E). Free-running periods of pdf > 4x t-PDF + TnTLC flies are also shorter than pdf > 4x t-SCR + TnTLC flies and longer than pdf > 4x t-PDF flies (Figure 6C, 6F), suggesting anadditional contribution to rhythm acceleration by classical neurotransmitter output inducedby PDFR autoreceptor activation. These results indicate a key role for PDFR autoreceptor-regulated co-release of PDF and classical neurotransmitter by LNvs in the daily allocation ofcircadian activity.

DiscussionStudies of the Drosophila circadian control circuit over the past decade have revealed acritical role for neuropeptide signaling between PDF secreting LNvs and PDF-negative

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PDFR-expressing clock neurons for generation and maintenance of robust circadian activityrhythms. While PDF-secreting LNvs also express PDFR, little is known about the functionalsignificance of these peptide autoreceptors, other than enhancing the robustness of free-running rhythms in DD (Lear et al., 2009). Here we show that PDFR autoreceptor activationin LNvs shifts the balance of daily circadian activity to the morning from the evening byengaging the Gα,s-cAMP pathway and thereby modulating secretion of PDF and co-released classical synaptic neurotransmitter.

While PDFR expression can be detected in both the sLNvs and lLNvs (Im and Taghert,2010), our results suggest that PDFR-induced increased morningness is likely mediated bysLNvs rather than lLNvs. First, sLNvs respond robustly to bath-applied PDF with increasedcAMP whereas lLNvs do not (Shafer et al., 2008), and downregulating Gα,s activity orcAMP levels suppresses the behavioral effects of LNv PDFR activation (Figure 2). Second,PDFR autoreceptor activation in LNvs cell-autonomously depolarizes the sLNvs while notaffecting the lLNvs (Figure 4). Third, expression of t-PDF with the sLNv-specific R6-GAL4driver induces a more robust increase in morningness than with the lLNv-specific c929-GAL4 driver (Supplemental Figure 2). Nevertheless, these findings do not rule out somecontribution of PDFR autoreceptors expressed in lLNvs (Im et al., 2010) in the t-PDFinduced shift of the balance of daily activity from evening to morning. While PDFRactivation by bath-applied PDF does not induce a cAMP increase in lLNvs (Shafer et al.,2008), it is possible that lLNv PDFR couples to non-cAMP signaling pathways such as Ca2+

signaling (Mertens et al., 2005). However, the suppression of t-PDF induced increasedmorningness by disruption of Ga,s-cAMP signaling (Figure 2) indicates that anycontribution of lLNv PDFR activation occurs upstream of the sLNvs.

We demonstrate that increased morningness induced by PDFR activation in LNvs requiresnot only PDF outputs (as expected from prior studies) but also classical synapticneurotransmitter co-release by the LNvs (Figure 6). Other behaviors such as feeding alsoemploy multiple co-released intercellular signals with different dynamics, with one signalinstructing the behavior and the other modulating the sensitivity to the instructive signal(Root et al., 2011). There is also evidence that neuropeptide and inhibitory synapticneurotransmitter co-secretion underlies unique behavioral adaptations (Tan and Bullock,2008). Determining the identity of the co-released classical neurotransmitter required forPDFR autoreceptor-mediated increased morningness will provide insight into the questionhow sLNv outputs influence downstream circadian clock neurons to determine daily activityrhythms.

What is the physiological significance of the increased morningness induced by PDFRactivation sLNvs? One clue is that this PDFR autoreceptor-induced increased morningnessmimics the behavioral response of flies to summer-like lighting conditions, where morningactivity is increased and/or phase advanced (Majercak et al., 1999; Stoleru et al., 2007;Zhang et al., 2010b). This suggests that PDF signals to LNvs might be regulated by lightconditions. Indeed, lLNvs are highly sensitive to light (Fogle et al., 2011; Sheeba et al.,2008a), contact sLNv post-synaptic sites (Helfrich-Forster et al., 2007), promote phase-advances to morning light (Shang et al., 2008), and their PDF secretion is important forentrainment by light (Cusumano et al., 2009). Thus, the lLNvs are well-situated to transferilluminance information to the sLNvs via PDF to regulate allocation of circadian activity andphase as photoperiod changes throughout the year. Consistent with this hypothesis,hyperexcitation of lLNvs by expression of NaChBac (low threshold bacterial voltage-gatedNa2+ channel) increases circadian morning activity (Supplemental Figure 3). This leads tothe model of a homotypic PDF relay circuit, where summer conditions with lengthened andbrighter dawn increase PDF secretion by lLNvs, thus increasing PDFR activation in sLNvs,thereby adaptively adjusting the allocation of circadian activity between morning and

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evening (Figure 7). Interestingly, a recent study has shown a strong temperature dependenceof the timing of morning activity in natural light and temperature conditions and aninduction of a prominent daytime activity bout at very high temperatures (Vanin et al.,2012), suggesting that temperature also regulates the distribution of daily activity (Zhang etal., 2010b). It is interesting to speculate that this temperature modulation of daily activityallocation relies on PDFR autoreceptors and the intra- and inter-celluar signaling pathwayswe have elucidated here.

There are many parallels between our model for PDFR autoreceptor signaling in theDrosophila circadian control network and VIP signaling in the mammalian circadiannetwork of the SCN. They include the fact that VIP expressing cells in the SCN areinnervated by the visual system (Tanaka et al., 1993) and that VIP secretion is stimulated bylight (Francl et al., 2010). Moreover, there is evidence that VIP signals modulate SCNtranscriptional responses to light (Dragich et al., 2010), mediate light-dependent phase-shiftsof circadian locomotor rhythms (Colwell et al., 2003; Piggins et al., 1995), and controllocomotor activity levels (Harmar et al., 2002). About 30% of all SCN VIP cells alsoexpress VIPR (Kallo et al., 2004), suggesting that VIPR autoreceptor signaling in the SCNplays a similar role to that of PDFR in modulating VIP/classical neurotransmitter co-release(Moore et al., 2002). Intriguingly, the possibility of mammalian VIPR autoreceptor signalinginfluencing light-dependent circadian behavior can be tested using the GPI-tethered peptidestrategy we employed here in the fly. VIPR is a class B1 G protein coupled receptor (GPCR)(Dickson and Finlayson, 2009), and we have previously shown the GPI-tethered peptidedesign we developed in the context of the fly is generalizable to mammalian class B1 GPCRligands (Fortin et al., 2009). Indeed, we have tested GPI-tethered VIP (t-VIP) in vitro inheterologous cells and found that it is a potent activator of VIPR (data not shown). Byexpressing t-VIP using the VIP promoter, analogous to our expression of t-PDF using thepdf promoter, one can test whether VIPR autoreceptor signaling modulates VIP-dependentcircadian behaviors such as arousal and phase shifts. Further studies of this nature willelucidate the cellular mechanisms by which peptide autoreceptor signaling modulates sleep/wake and activity control circuits as well as reveal evolutionarily conserved principles forthe modulation of daily behavioral rhythms by the environment.

MethodsFly strains and Crosses

All crosses and behavioral experiments were performed at 25°C. UAS-t-PDF (described asUAS-t-PDF-ML in (Choi et al., 2009)), UAS-t-SCR (described as UAS-t-PDF-SCR in (Choiet al., 2009)) and UAS-t-DH31 are as described previously (Choi et al., 2009). Twoindependent insertions of each transgene were combined via homologous recombination togenerate second and third chromosomes that contain two copies of each transgene. The 2xUAS-t-PDF or 2x UAS-t-SCR flies were further crossed to each other and the pdf-GAL4(II)driver (Renn et al., 1999) to generate flies with up to six copies of either transgene with asingle copy of pdf-GAL4 (pdf-GAL4/2x UAS-t-PDF; 2x UAS-t-PDF/2x UAS-t-PDF or pdf-GAL4/2x UAS-t-SCR; 2x UAS-t-SCR/2x UAS-t-SCR).

For sLNv PDFR activation in pdf01 null background, a pdf-GAL4 insertion on the ×chromosome was used. pdf-GAL4(X) was combined with 1x UAS-t-PDF or 2x UAS-t-PDFon the second chromosome and the pdf01-null third chromosome. Homozygous males with atotal of 2 or 4 copies of t-PDF in the pdf-null background were used for behavioral assays(pdf-GAL4; 1x or 2x t-PDF; pdf01).

UAS-TnTLC is from Brian McCabe, UAS-Gas[GTP] gain of function mutant (Wolfgang etal., 1996), UAS-Gas-11 peptide inhibitor (Yao and Carlson, 2010) is from John Carlson,

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pde8EY enhancer trap line is from Justin Blau. Gas RNAi lines are obtained fromDrosophila Genomics Resource Center and Vienna Drosophila Stock Center (stocknumbers: 24958 and 105485).

Behavioral AssaysLocomotor activity monitoring—Individual 3–5 day old male flies were placed inlocomotor activity monitor tubes and were entrained in 25°C, 12hr: 12hr light:dark conditions (LD) for at least 5 days and then released into free running conditions of constantdarkness (DD). All flies were monitored simultaneously with its respective controls. Theautomated TriKinetics (Waltham, MA) infrared beam-crossing monitor system was used toassay locomotor activity. 20 minute bin-size double-plotted actograms and Lomb-Scargleperiodograms for assessment of free running period were generated using ActimetricsClocklab software (Wilmette, IL), running on MATLAB. 30 minute bin averaged activityhistograms in LD were generated by averaging 30 minute bin activity profiles over 4 days inLD, then averaging across animals. 30 minute bin first day of DD averaged activityhistograms were generated by averaging the 30 minute bin activity profiles across animals.

Statistics of locomotor activity—Morning anticipatory activity in LD was defined asthe average cumulative activity 3 hours prior to lights on (ZT22-ZT0) over four days in LDand averaged across animals within the same genotype. Morning anticipation phase score inLD was calculated to detect the presence incremental activity before lights-on, which is theratio of activity 3 hours prior to lights-on to the activity 6 hours prior to lights-on(Harrisinghet al., 2007).

The morning peak, evening peak and morning ratio in the first day of DD for each animalwas determined from moving averages of the average activities. From this smoothenedactivity profile, we determined the start of a peak as the starting point of a continuousincrease of activity towards the peak maximum, allowing 1 step decrease in this duration.The end of a peak was determined as the end of a continuous decrease of activity from thepeak maximum, allowing 1 step increase in this duration. Morning peak and evening peakwere calculated as the sum of the activity in this duration, either in subjective dawn orsubjective dusk, in individual animals and averaged across animals. Morning ratio wascalculated as the activity during the morning peak over the activity during the evening peak,and averaged across animals. In a few animals, the evening activity was so low that thecalculated morning ratio exceeded 20, and such animals were assigned a morning ratio of20.

Free-running periods for each animal were determined using Lomb-Scargle periodogramsfor 11 days in constant darkness, starting on the first day of DD, and averaged for eachgenotype. Average actograms representing free-running behavior were generated usingActimetrics Clocklab software (Wilmette, IL), where 20 minute activities normalized to thedaily activity of each animal is averaged across multiple animals of each genotype.

Statistical significance of the genotype-dependent effects on free-running period, morningactivity and morning ratio were tested with One-way ANOVA and Bonferroni All-PairwiseMultiple Comparison Test with α = 0.05.

ImmunocytochemistryAdult male fly brains were dissected and processed for immunofluorescence with mouseanti-PDF (1:50) primary antibody and Cy2-conjugated anti-mouse secondary antibody(1:200), as described previously (citation). Anti-PDF fluorescence in the dorsal brain regionwas collected using a charge-coupled device (CCD) camera mounted on a Zeiss Axiophot

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epifluorescent photomicroscope with 64X optical lens and used for analysis. Average pixelvalue from the background was subtracted from the threshold-selected pixels from the dorsalPDF+ projections of that image. Integrated pixel values of the threshold-selectedbackground subtracted images were normalized to the greatest integrated pixel value withineach experiment.

Clock neuron electrophysiologyAdult Drosophila Whole-Brain Explant Preparation—Flies were maintained at 25°Cin a 12hr/12hr light/ dark (LD) cycle. 7–10 days post-eclosion males of the genotypes, pdf-GAL4, UAS-DsRed/ 2x t-PDF; 2x t-PDF (6x t-PDF) or pdf-GAL4, UAS-DsRed/ 2x t-SCR;2x t-SCR (6x t-SCR) were dissected at ZT21.5–22.5 for electrophysiological recordings.These flies express red fluorescent protein, DsRed, solely in ventral lateral clock neurons(LNV). Whole-cell recordings on small LNV (sLNVs) of fly brain explants were performedas described (Cao and Nitabach, 2008; Wu et al., 2008a)), and all recordings were donebetween ZT 22–23. Briefly, the fly brains were dissected in external recording solution,which consisted of (in mM): 101 NaCl, 3 KCl, 1 CaCl2, 4 MgCl2, 1.25 NaH2PO4, 5Glucose, 20.7 NaHCO3, pH 7.2 with osmolarity of 250 mmol/kg. The brain was placedanterior side up, secured in a recording chamber with a mammalian brain slice “harp”holder, and was continuously perfused with external solution bubbled with 95% O2/5% CO2at room temperature (22 °C). For tetrodotoxin (TTX) experiments, TTX (final concentration:2µM) was added to the perfusion solution. sLNVs and lLNvs were visualized by DsRedfluorescence and distinguished by their sizes, and subsequently, the immediate areasurrounding the sLNV or lLNv cell bodies was enzymatically digested with focal applicationof protease XIV (2 mg/ml, Sigma-Aldrich).

Whole Cell Patch-Clamp Electrophysiology—The voltage-gated and ligand-gatedconductances of sLNvs – as for many adult Drosophila brain neurons – are electrotonicallydistant from the recording site at the soma, thus preventing voltage-clamp of thoseconductances. Accordingly, electrical properties of the sLNvs were assayed using currentclamp, with the caveat that electrical properties measured at the soma reflect more distalproperties filtered by the neuron’s cable properties.

Whole-cell recordings were performed using borosilicate standard wall capillary glasspipettes (Sutter Instrument Company, Novato, CA). Recording pipettes were filled withinternal solution consisting of (in mM): 102 potassium gluconate, 17 NaCl, 0.085 CaCl2, 4Mg-ATP, 0.5 Na-GTP, 0.94 EGTA and 8.5 HEPES, pH 7.2 and osmolarity of 235 mmol/kg.The resistance of filled pipettes was 8–12 MΩ. Gigaohm seals were achieved beforebreaking in to whole-cell configuration in voltage-clamp mode. To confirm maintenance ofa good seal and absence of damage to the cell, a 40 mV hyperpolarizing pulse was imposedon each cell while in whole-cell voltage-clamp mode from a holding potential of −80 mV.Only if the resulting inward leak current was less than −100 pA was that cell used forsubsequent measurements.

After switching from voltage-clamp to current-clamp mode, resting membrane potential(RMP) was determined after stabilization of the membrane potential (5 min after thetransition). For cells with oscillating membrane potential, RMP was defined at the trough ofthe oscillation. Spontaneous activity was observed over the 10 min period after the transitionto current-clamp configuration. All cells included in this study were capable of firing actionpotentials when injected with positive current. Only one sLNv or lLNv per animal wasrecorded.

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Data Acquisition and Analysis—Signals were measured using a Multiclamp 700Bamplifier (Molecular Devices/Axon Instruments, Sunnyvale, CA) and a Digidata 1440Aanalog/digital converter (Molecular Devices/Axon Instruments). The inward leak currentand RMP were measured in Clampfit, which is part of the pClamp 10 software package.Cross-correlational analysis was also conducted in Clampfit on the last 100 s of the first 5minutes after the transition from voltage-clamp to current-clamp mode. The signal was firstfiltered by a lowpass Gaussian filter with a −3 db cut off of 5 Hz. Cross-correlation was thenrun and the correlation was defined as the amplitude of the peak of correlation. If no peakwas observed, the recording was assigned a correlation value of the minimum correlationfound within the experiment. Since correlation values for some samples were not measuredbut assigned at a minimum, the non-parametric Mann-Whitney U test was used to determinethe statistical significance of the differences in correlation. T-test was used to determine thestatistical significance of the RMP.

Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.

AcknowledgmentsWe thank P. Taghert, L. Griffith, J. Blau, J. Carlson, and B. McCabe for fly stocks; M. Kunst for figure graphicsand comments on the manuscript; M. Hughes and D. Raccuglia for advice on statistics and comments on themanuscript; and J. Duah, C. Drucker and G. Barnett for assistance in fly maintenance and assistance in behavioralexperiments. Work in the laboratory of M.N.N. is supported in part by the National Institute of NeurologicalDisorders and Stroke, National Institutes of Health (NIH) (R01NS055035, R01NS058443, R21NS058330) andNational Institute of General Medical Sciences (R01GM098931), National Institutes of Health (NIH). C.C. issupported in part by the National Institute of General Medical Sciencs, NIH (T32GM07527).

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Highlights

• Activation of PDF receptors in sLNv clock neurons increases circadian morningactivity.

• This phenotype is associated with sLNv depolarization and increased PDFsecretion at dawn.

• This phenotype requires the Gα,s-cAMP pathway and co-secretion of PDF andneurotransmitters.

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Figure 1. Activation of PDFR autoreceptors in LNvs increases morningness and accelerates free-running rhythms(A) Average LD locomotor histograms. Filled bars and unfilled bars represent 30 minute binactivity during light phase (ZT0-ZT12) and dark phase (ZT12-ZT0) respectively. Fliesexpressing t-PDF show greater morning anticipation with increasing numbers of UAS-t-PDFtransgenes. Negative controls are flies expressing the inert t-PDF isoform, t-SCR, from sixUAS-t-SCR transgenes (pdf > 6x t-SCR). See Supplemental Table 1 for Ns.(B) Average locomotor histograms on the first day of DD (DD1, ZT15-CT18) for the sameflies as in (A). The size of morning peak relative to the evening peak increases withincreasing dose of t-PDF. See Supplemental Table 1 for Ns.

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(C) Averaged actograms for 11 days in constant darkness (DD1 – DD11) for the same fliesas in (A). Black bars indicate subjective night and grey bars indicate subjective day. SeeSupplemental Table 1 for Ns.(D) LNv PDFR activation increases morning anticipatory activity during the 3 hours prior tolights-on (ZT21-ZT0) in LD in a dose-dependent manner. One-Way ANOVA indicatessignificant differences between genotypes (P < 0.0001), and Bonferroni All-PairwiseMultiple-Comparison test (α = 0.05) reveals significantly greater morning anticipatoryactivity in pdf > 4x t-PDF than in controls and pdf > 2x t-PDF flies, and even greatermorning anticipatory activity in pdf > 6x t-PDF flies.(E) LNv PDFR activation shifts circadian activity to the subjective morning, as revealed bycomparisons of morning ratios, which is defined as the ratio of total activity during themorning peak to the total activity peak during the evening peak (see Methods). One-WayANOVA indicate significant differences between genotypes (P < 0.0001), and BonferroniAll-Pairwise Multiple-Comparison Test (α = 0.05) reveal significantly greater morningnessin pdf > 6x t-PDF flies.(F) Average free-running periods during the first 11 days in DD. Free-running rhythmsaccelerate with increasing t-PDF copy number. One-way ANOVA indicate significantdifference among genotypes (P < 0.0001), and Bonferroni All-Pairwise Multiple-Comparison Test (α = 0.05) indicate significantly shorter free-running periods in pdf > 4x t-PDF and pdf > 6x t-PDF compared to the control.(D–G) Genotype labels at the top of each bar indicate significant differences from, a: pdf >6x t-SCR, b: pdf > 2x t-PDF, c: pdf > 4x t-PDF, d: pdf > t-PDF, as detected by BonferroniAll-Pairwise Multiple Comparison Test (α = 0.05). Bars and error bars indicate mean andstandard errors.

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Figure 2. PDFR autoreceptor activation increases morningness through Gα,s-cAMP-pathway(A) Inhibition of Gα,s or phosphodiesterase-8 overexpression in LNvs suppress LNv PDFRactivation induced morningness increase. Line graphs represent average LD and DD1activity of flies expressing Gα,s peptide inhibitor (Gα,s-11) or phosphodiesterase 8 (PDE8)with either 4x t-PDF or 4x t-SCR. One-way ANOVA indicate significant differences inmorning anticipatory activity (P < 0.0001) and morning ratio (P < 0.0001) among genotypes.Increase in morning anticipatory activity induced by t-PDF expression is partiallysuppressed by Gα,s-11 and fully suppressed by PDE8 co-expression. t-PDF expressioninduced increase in morning ratio is fully suppressed by Gα,s-11 or PDE8 co-expression.See Supplemental Table 1 for Ns.

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(B) RNAi knockdown of Gα,s suppress morningness increase induced by PDFR activationin LNvs. Line graphs represent average LD and DD1 activity of flies expressing Dicer,transgenes for RNAi knockdown of Gα,s (Gαs RNAi VDRC#24958 and Gαs RNAiDGRC) and either 4x t-PDF or 4x t-SCR. One-way ANOVA indicate significant differencesin morning anticipatory activity (P < 0.0001) and morning ratio (P < 0.0001) amonggenotypes. Increase in morning anticipatory activity and morning ratio induced by t-PDFexpression is fully suppressed by Gαs RNAi VDRC#24958 co-expression. Co-expression ofGa,s RNAi DGRC also fully suppresses the t-PDF induced morning ratio increase, but onlypartially suppresses morning anticipatory activity increase, likely reflecting the lesscomplete Gα,s knockdown by Gαs RNAi DGRC than by Gαs RNAi VDRC#24958. SeeSupplemental Table 1 for Ns.(C) Activation of Gα,s pathway by expression of constitutively active Gα,s mutant(Gα,s[GTP]) increases morningness with or without PDFR activation. Line graphs representaverage LD and DD1 activity of flies expressing Gα,s[GTP] with either 4x t-PDF or 4x t-SCR. One-way ANOVA indicate significant differences in morning anticipatory activity (P< 0.01) and morning ratio (P < 0.0001) among genotypes. Either t-PDF or Gα,s[GTP]expression significantly increased morning anticipatory activity or morning ratio, without noadditive effects of t-PDF and Gα,s[GTP] co-expression on morningness. See SupplementalTable 1 for Ns.(A–C) Yellow indicate day, blue indicate night and subjective night and light blue indicatessubjective day. n.s., not significant; *, • = 0.05; **, • = 0.01; ***, • = 0.001; significantdifference detected by Bonferroni All-Pairwise Multiple-Comparison Test. Bars and errorbars indicate means and standard errors.

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Figure 3. Activation of DH31 receptors in LNvs does not increase morningness(A–C) Average LD (A) and DD1 (B) locomotor histograms and averaged actograms (C) offlies expressing t-DH31 in LNvs from either of two different combinations of sixindependent UAS-t-DH31 transgenes (6x t-DH31(a) or 6x t-DH31(b)). See SupplementalTable 1 for Ns.(D) Mild decrease of morning anticipatory activity by LNv DH31R activation. One-wayANOVA reveals significant differences (P < 0.05), with only the pdf > 6x t-DH31(b)combination exhibiting significantly less morning anticipatory activity.

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(E) Mild decrease in morningness by LNv DH31R activation. One-way ANOVA revealssignificant differences (P < 0.001) with only the pdf > 6x t-DH31(a) combination exhibitingsignificantly less morning ratio.(F) Free-running rhythms in DD are slightly shortened with LNv DH31R activation. One-way ANOVA, p < 0.0001.(D–F) n.s., not significant; *, • = 0.05; **, • = 0.01; ***, • = 0.001; significant differencedetected by Bonferroni All-Pairwise Multiple-Comparison Test. Bars and error bars indicatemeans and standard errors.

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Figure 4. Activation of PDFR autoreceptors in LNvs specifically depolarizes sLNvs(A) Representative 5 second traces of whole-cell current-clamp recordings of sLNv andlLNv clock neurons in pdf > 6x t-SCR and pdf > 6x t-PDF flies. Recordings were conductedat ZT22–23.(B) Average resting membrane potential (RMP) of sLNv and lLNvs in pdf > 6x t-SCR andpdf > 6x t-PDF flies. t-PDF expression specifically depolarizes sLNvs, with no effect onlLNvs. There are no effects on lLNv RMP, but sLNvs are significantly depolarized with t-PDF expression. *, p < 0.03, n.s., not significant, unpaired t-test.(C) Autocorrelation analysis of RMP oscillation of sLNv and lLNv cells in pdf > 6x t-SCRand pdf > 6x t-PDF flies. sLNv RMP oscillation is completely suppressed by t-PDFexpression, while lLNvs are not affected. *, p < 0.02, n.s., not significant, Mann-Whitney UTest, See Methods.(D) Representative 5 second traces of whole-cell current-clamp recordings of sLNv andlLNv clock neurons in pdf > 6x t-SCR and pdf > 6x t-PDF flies in the presence of bath TTX.Recordings were conducted at ZT22–23. Note the absence of regular RMP oscillations andaction potentials.(E) Average resting membrane potentials (RMP) of sLNvs and lLNvs in pdf > 6x t-SCR andpdf > 6x t-PDF flies in the presence of bath TTX. sLNvs are significantly depolarized by t-PDF expression, while lLNvs are not affected. *, p < 0.01, n.s., not significant, unpaired t-test.(B, C, E) Bars and error bars indicate mean and standard errors. Number of recorded cells isindicated in the bars.

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Figure 5. PDFR activation in PDF-negative clock neurons is necessary and sufficient for morningactivity(A) PDFR activation in LNvs without PDF secretion from LNvs has no effect on circadianlocomotor activity. Average LD and DD1 activity histograms of pdf01 null-mutant fliesexpressing 4x t-SCR, two transgene combinations of 2x t-PDF or 4x t-PDF. In LD,stereotypical lack of morning anticipation and phase-advanced evening anticipation of pdf01

null-mutant flies is seen for all genotypes with no differences in their circadian locomotorprofiles. Morning anticipation phase scores indicate absent morning anticipation (phasescore = 0.5) in all genotypes (One-way ANOVA, P > 0.85). In DD1, circadian activity peaksare severely dampened or absent in all genotypes. Bars and error bars indicate means andstandard errors.

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(B) PDFR activation in PDF-negative clock neurons is sufficient for circadian activitypeaks. Average LD and DD1 activity histograms of pdf01 null-mutant flies expressing t-SCRor t-PDF from one or two transgenes in PDF-negative clock neurons, using the combinationof tim-GAL4 driver active in all clock neurons and pdf-GAL80 to suppress GAL4 activity inPDF-positive LNvs. t-PDF expression in PDF-negative neurons of pdf01 null-mutant fliesrescues robust morning anticipatory activity in LD as revealed by calculating morninganticipation phase scores. One-way ANOVA indicate significant differences among t-PDFexpressing pdf01 null-mutants and their t-SCR expressing pdf01 null-mutant controls (P >0.0001), and Bonferroni All-Pairwise Multiple-Comparison Test (*, α = 0.05) revealssignificantly greater morning anticipation in t-PDF expressing pdf01 null-mutants comparedto t-SCR expressing pdf01 null-mutant controls. In DD1, t-PDF expression in PDF-negativeclock neurons of pdf01 null-mutant also rescues robust morning and evening circadianactivity peaks that are severely damped in t-SCR-expressing controls. Bars and error barsindicate means and standard errors.(C) Representative images of anti-PDF immunohistochemistry and average normalized PDFlevels in sLNv dorsal terminals of pdf > 6x t-PDF and pdf > 6x t-SCR flies at ZT22. Bargraph shows pooled results from four independent experiments. PDF accumulation in sLNvdorsal terminals is greater in pdf > 6x t-PDF flies than in pdf > 6x t-SCR controls. ***, P <0.0001, un-paired t-test. Bars and error bars indicate means and standard errors. Ns areindicated in the bars.

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Figure 6. Blocking classical synaptic transmission suppresses increased morningness induced byLNv PDFR autoreceptor activation(A–C) Average LD (A) and DD1 (B) locomotor histograms and averaged actograms (C) offlies co-expressing 4x t-PDF or 4x t-SCR with tetanus toxin light chain (TnTLC). Increasedmorningness induced by PDFR activation in LNvs is prevented when classical synaptictransmission is blocked by TnTLC co-expression. See Supplemental Table 1 for Ns.(D) TnTLC co-expression strongly suppresses the increase in morning anticipatory activityinduced by LNv PDFR activation. One-way ANOVA indicates significant differences inmorning ratio between genotypes (P < 0.0001), and Bonferroni All-Pairwise Multiple-

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Comparison Test reveals significantly less morning anticipatory activity in pdf > 4x t-PDF +TnTLC flies than in pdf > 4x t-PDF flies.(E) TnTLC co-expression completely inhibits the morningness increase induced by LNvPDFR activation. One-way ANOVA indicates significant differences in morning ratiobetween genotypes (P < 0.001), and Bonferroni All-Pairwise Multiple-Comparison Testreveals significant increase in morning ratio by t-PDF expression and no significant changein morning ratio by t-PDF expression when TnTLC is co-expressed.(F) TnTLC co-expression partially suppresses the period shortening effect of LNv PDFRactivation without affecting free-running period on its own. One-way ANOVA indicatessignificant differences in morning ratio between genotypes (p < 0.0001).(D–F) **, p<0.01, ***, p < 0.001, significant differences detected by Bonferroni All-Pairwise Multiple-Comparison Test. n.s., not significant. Bars and error bars indicate meansand standard errors.

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Figure 7. Model of PDFR autoreceptor signaling in LNvsPDFR in sLNvs activates Gα,s and stimulates cAMP synthesis by adenylyl cyclase. Thesubsequent increase in intracellular cAMP leads to membrane depolarization, increased PDFsecretion and altered secretion of an unknown neurotransmitter (NT). Increased PDF andneurotransmitter output from sLNvs induced by PDFR activation acts on secretion acts ondorsal clock neurons to increase circadian morning activity. PDFR in sLNvs is activated byincreased PDF secretion by lLNvs in response to light.

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Drosophila NIH Public Access from PDF Neurons Determines ... · (pdf > 2x t-PDF, pdf > 4x t-PDF or pdf > 6x t-PDF) for examination of dose-dependent effects of PDFR activation. Flies