Differentially Timed Extracellular Signals Synchronize Pacemaker Neuron Clocks

Circadian pacemaker neurons in Drosophila are regulated by two synchronizing signals that are released at opposite times of day, generating a rhythm in intracellular cyclic AMP.


Introduction
Coordinated neuronal activity is vital for neural networks to regulate complex processes such as behavior.Synchrony can be studied at the microsecond level by measuring neuronal activity, with synchronous activity often achieved via gap junctions that electrically couple neurons [1].The circadian system offers an unusual opportunity to study synchrony over a much longer timeframe as circadian pacemaker neurons have molecular clocks that oscillate with 24 hour periods.These endogenous clocks drive daily rhythms in pacemaker neuron electrical activity and allow organisms to anticipate environmental transitions such as sunrise and sunset [2].Although the molecular basis of the circadian clock is well established, how individual clock neurons remain synchronized is much less well understood.Synchrony is essential in the circadian system as the accuracy of individual clocks would be meaningless if they were desynchronized.Coordinated molecular clocks presumably ensure that an animal has a single internal representation of time.
In mammals, the primary circadian pacemaker in the suprachiasmatic nucleus (SCN) consists of ventral ''core'' and dorsal ''shell'' regions of clock neurons.Although SCN clock neurons exhibit 24 hour oscillations of clock proteins, anatomically distinct neurons oscillate with different phases (reviewed by [3]).Oscillations within different SCN neurons are coupled through cyclic AMP (cAMP) and Ca 2+ -dependent mechanisms, promoting synchrony and increasing the amplitude of individual oscillators compared to non-SCN clock neurons (reviewed by [4]).Synchronizing the different phases of SCN oscillations requires RGS16, which is rhythmically expressed and inactivates the G-protein Gai to increase cAMP levels in the SCN in a time-dependent manner [5].
The Drosophila clock circuit also contains distinct groups of neurons including the small ventral Lateral Neurons (s-LN v s) that communicate with a subset of dorsal Lateral Neurons (LN d s) and Dorsal clock neurons (DNs) to generate bimodal locomotor activity rhythms in light:dark (LD) cycles [6,7].s-LN v s are often called master pacemaker neurons as they set the period for most of the clock network in constant darkness (DD) [8].However, robust behavioral rhythms in DD require LN v and non-LN v neurons to signal at different times of day [9].Different groups of clock neurons also respond differently to environmental stimuli, such as day length or temperature [10,11], leading to a network view of the clock where different clock neuron groups process information and communicate to keep time for an individual animal [12].
The mammalian neuropeptide VIP and the Drosophila neuropeptide PDF are found in subsets of clock neurons: ventral core SCN neurons in mice and LN v s in flies [3,13].VIP and PDF are both required for robust behavioral rhythms, the maintenance of stable phase relationships between different groups of clock neurons, and synchronized molecular clock oscillations within individual groups of clock neurons [8,[14][15][16][17].The PDF receptor (PdfR) and VIP receptor VPAC2R are also required for robust rhythms of behavior, and they both activate Gas to increase cAMP levels, indicating a conserved mode of action [18][19][20][21].However, the precise mechanisms by which signaling across the clock circuit promotes synchronous clock oscillations remain unclear.
We used Drosophila to understand how circadian networks are synchronized, taking advantage of the exquisite precision with which individual groups of clock neurons can be manipulated in flies and the variety of genetic tools available.We made extensive use of the minimal larval clock circuit, which has only nine clock neurons per brain lobe, including four PDF-expressing LN v s that display synchronous clock protein oscillations in constant darkness (DD).These rhythms require the transcription factors Clock (CLK) and Cycle (CYC) that activate period (per) and timeless (tim) transcription.PER and TIM proteins dimerize, enter the nucleus, and then inhibit CLK/CYC activity.This represses expression of per, tim, and other CLK/CYC targets, including vrille and Par Domain Protein 1 (Pdp1), that in turn feed back to regulate Clk expression (reviewed by [22]).One entire cycle takes 24 hours.
Synchronized LN v oscillations in adult flies require PDF, as s-LN v clocks become desynchronized in Pdf 01 null mutants after 6-9 days in constant darkness [17].Here we show that LN v synchrony in DD is a very active process, as desynchrony can be detected as early as 3 hours into the first subjective morning in Pdf 01 mutant larvae.We show that synchronized LN v clocks require two distinct signals: a neuropeptide signal (PDF) received around dawn via PdfR and a neurotransmitter signal (glutamate) received from DN 1 s around dusk via the metabotropic glutamate receptor (mGluRA).
Surprisingly, simultaneously reducing expression of Pdfr and mGluRA in LN v s severely dampened TIM protein oscillations and blocked larval behavioral rhythms.Thus, oscillations of core clock proteins within pacemaker neurons require signals from other clock neurons.PdfR and mGluRA are GPCRs, and we show that daily oscillations in LN v cAMP levels depend on their receiving PDF and glutamate.Because cAMP has previously been shown to be a molecular clock component in mammals [23], our data provide a mechanism for how extracellular signals impact molecular oscillations and neuronal synchrony.We extend these findings to adult flies and show that PdfR and mGluRA are required to maintain synchronized high-amplitude TIM oscillations in s-LN v s.In adults, desynchronized s-LN v molecular clocks are associated with noisy behavioral rhythms, including delayed onset of sleep and increased nighttime activity.
Our data reveal a surprising degree of conservation in the mechanisms promoting synchronous clock oscillations in the mammalian SCN and Drosophila LN v s.This mirrors the conserved molecular basis of mammalian and Drosophila clocks and indicates that studying the simple Drosophila circadian neural circuit will help understand the more complex mammalian circadian system.

PDF Signaling Synchronizes Larval LN v s
The four PDF-expressing LN v s in each larval brain lobe are precursors of adult s-LN v s.Molecular clock oscillations in larval LN v s are normally tightly synchronized, oscillating in phase with each other so that TIM and PDP1 clock proteins are detectable in all four LN v s at CT21 and undetectable in all four LN v s 6 hours later at CT3 (Figure 1A) (CT, Circadian time, hours in constant darkness).
Because PER protein rhythms in adult s-LN v s become desynchronized in Pdf 01 null mutants in DD [17], we first tested whether PDF is required to synchronize larval LN v molecular clocks.We measured TIM protein levels instead of PER with the rationale that TIM's shorter half-life [24,25] would allow us to detect desymchrony earlier in DD.
To visualize LN v s in Pdf 01 mutants, we used the Gal4/UAS system [26] to express GFP in LN v s using the Pdf-Gal4 driver.We measured TIM levels in LN v s isolated at CT9, CT15, and CT21 on the second day in DD and at CT3 on day 3. TIM continues to oscillate in Pdf 01 mutants, indicating that the molecular clocks in their LN v s are functional (Figure 1A-B).However, the amplitude of TIM rhythms in Pdf 01 mutants was reduced compared to controls (Figure 1B), as expected from the reduced amplitude tim RNA oscillations in Pdf 01 adult flies [27].Closer inspection identified a mixture of TIM-positive and TIM-negative LN v s in a single brain lobe at CT15, 21, and 3 in Pdf 01 mutants (Figures 1D and S1A; see Materials and Methods), which we term desynchronized.Elevated

Author Summary
Circadian molecular clocks are essential for daily cycles in animal behavior and we have a good understanding of how these clocks work in individual pacemaker neurons.However, the accuracy of these individual clocks is meaningless unless they are synchronized with one another.In this study we show that synchronizing the principal pacemaker LN v neurons in Drosophila larvae require two extracellular signals that are received at opposite times of day: namely, the neuropeptide PDF released from LN v s themselves at dawn and glutamate released from dorsal clock neurons at dusk.LN v s perceive both PDF and glutamate via G-protein coupled receptors that increase or decrease intracellular cAMP, respectively.The alternating phases of PDF and glutamate release generate oscillations in intracellular cyclic AMP.In addition to maintaining synchrony between LN v s, this rhythm is also required for molecular clock oscillations in individual larval LN v s.We show that disruption of PDF and glutamate signaling also reduces synchrony in adult LN v s.This impairs the oscillations of clock proteins and flies have delayed onset of sleep.Our data highlight the importance of intercellular signaling in ensuring synchrony between clock neurons within the circadian network.Our findings help extend the conservation of clock properties between Drosophila and mammals beyond clock genes to include clock circuitry.S1.
desynchrony likely accounts for the significantly lower average TIM levels in Pdf 01 LN v s at CT15 and 21 than in controls (Figure 1B), in agreement with previous reports [17,27].
We also quantified the variability within individual LN v clusters by calculating the standard deviation in TIM levels across a single cluster.Figure 1F shows the distribution of standard deviations in TIM levels for each control or Pdf 01 LN v cluster at CT3 and CT9.We chose CT3 because desynchronized LN v clusters were only rarely found at this timepoint in control larvae.In contrast, TIM was detected in one, two, or three of the four LN v s in 50% of Pdf 01 LN v clusters at CT3 (n = 20; Figures 1D and S1A and Table S1).In subsequent experiments we used the presence of TIM in a subset of LN v s at CT3 to indicate that an LN v cluster had lost its normal coherent phase relationship and had become desynchronized, even if we did not observe desynchrony at other time points.
Our data show significantly more variability in TIM levels within an LN v cluster in Pdf 01 mutants than in control larvae at CT3 (Figure 1F), reflecting desynchronized molecular clocks in Pdf 01 LN v s.No significant increase in standard deviation was observed in Pdf 01 mutants compared to controls at CT9 (Figure 1F).Indeed, the low TIM levels at CT9 indicate that Pdf 01 LN v molecular clocks still oscillate as shown previously [17,27].
Because PDF signals via PdfR, we next tested whether the synchrony of larval LN v molecular clocks is also altered in Pdfr mutants.Although overall TIM oscillations were similar between control and Pdfr han5304 (Pdfr han ) hypomorphs, we observed higher TIM levels at CT3 in Pdfr han than in control larvae (Figure 1A-B).As with Pdf 01 null mutants, this is because TIM was detected in 1-3 of the four LN v s in 48% of Pdfr han mutant LN v clusters at CT3 (Figures 1D and S1A).We found similar results for PDP1 (Figures 1A,C,E and S1B).In contrast, TIM or PDP1 expression was detected in ,5% of control LN v s at CT3 (n = 21; Figure 1D-E and Table S1).The standard deviations in TIM and PDP1 levels are significantly elevated at CT3 in Pdfr han mutants compared to controls (Figure 1F-G).Thus PdfR, like PDF, is required for LN v s to stay synchronized.
In contrast to Pdf 01 LN v s, Pdfr han mutants did not show many desynchronized LN v clusters at CT15 or CT21, and there was no corresponding reduction in the amplitude of TIM oscillations in Pdfr han mutants compared to control LN v s.This could be because Pdfr han is a hypomorph rather than a null allele and/or because type II GPCRs tend to be promiscuous, so receptors other than PdfR may also respond to PDF [28].
We also tested whether LN v molecular clocks required PDF to maintain synchrony under LD cycles.We measured TIM and PDP1 levels in control larvae and in Pdf 01 and Pdfr han mutants at ZT3, but detected no TIM or PDP1 expression in LN v s (Figure S1C).Thus, light overrides desynchrony in Pdf 01 and Pdfr han5304 mutants, with PDF signaling required for synchronous LN v clock oscillations only in DD.

PdfR Functions in Both LN v s and Other Clock Neurons to Synchronize LN v Clocks
Because adult and larval LN v s express Pdfr ( [29,30] and Figure S2C), the simplest model to explain how PDF promotes LN v synchrony would be that the four larval LN v s signal to synchronize each other via PDF and PdfR.However, Pdfr is also expressed in many non-LN v adult clock neurons [29] and in larval DN 1 s (Figure S2A,B).Thus PDF signaling to non-LN v s could also be required for LN v synchronization.We therefore used a Pdfr RNAi transgene [31] to reduce Pdfr levels in subsets of clock neurons to determine where PDF signaling is required for LN v synchronization.Expressing Pdfr RNAi in LN v s significantly reduced the cAMP response of LN v s to PDF, indicating that Pdfr RNAi likely reduces Pdfr expression (Figure S2C).UAS-Dicer-2 (UAS-Dcr-2) was coexpressed to increase RNAi efficacy in this and in all subsequent RNAi experiments unless otherwise stated, but is omitted from written genotypes in the text for simplicity.
We first targeted Pdfr RNAi to LN v s using Pdf-Gal4 (denoted as Pdf.).At CT3 on day 3 of DD, TIM staining revealed that 44% of Pdf.Pdfr RNAi larvae had desynchronized LN v s, whereas .93% of control LN v s were synchronized (Figures 2A and S3A and Table S1).The standard deviation in TIM levels was also significantly increased in Pdf.Pdfr RNAi larvae compared to controls at CT3 (Figure 2B).Similar results were observed for PDP1 at CT3 (Figure S3B and Table S1).
Next, Pdfr expression was reduced in all non-LN v clock neurons using the tim-Gal4; Pdf-Gal80 driver combination (tim; Pdf-Gal80.).We found that 44% of LN v s were desynchronized in tim; Pdf-Gal80.Pdfr RNAi larvae (Figure S3A and Table S1).This probably underestimates the level of defective TIM oscillations, as 16% of tim; Pdf-Gal80.Pdfr RNAi LN v clusters showed four LN v s expressing TIM at CT3, compared to only 6% of controls (Table S1).There is a corresponding increase in the standard deviation in TIM levels in tim; Pdf-Gal80.Pdfr RNAi LN v clusters compared to control LN v s (Figure 2B).Similar results were observed for PDP1 (Figure S3A-B).These data indicate that LN v synchrony depends on PdfR activity in both LN v and non-LN v clock neurons.

DN 1 s Synchronize Molecular Clock Oscillations in LN v s
The non-LN v clock neurons releasing the synchronizing signal could be the larval DN 1 s, the DN 2 s, or the fifth LN v .DN 1 s are the best candidates, as they project to LN v axonal termini and modulate LN v outputs by releasing glutamate to generate * p,0.05; ** p,0.01; *** p,0.001; **** p,0.0001.(A) Representative images of y w (Control, top panels), Pdf 01 mutants (middle), and Pdfr han mutants (bottom) stained for PDF or GFP (green), TIM (red), and PDP1 (blue).The lower panels for each genotype are the same images with the green channel removed and replaced by a dashed white line outlining the LN v s.Pdf 01 LN v s were identified via anti-GFP antibody staining of a UAS-GFP transgene driven by Pdf-Gal4, and PDP1 was not included in this experiment.(B) TIM immunostaining was quantified in Control (blue), Pdfr han (red), and Pdf 01 (green) LN v s on days 2 and 3 in DD.TIM oscillates in Pdfr han (ANOVA F 3,37 = 13.68,p,0.0001) and Pdf 01 (ANOVA F 3,56 = 16.80,p,0.0001) mutants.However, there is significantly more TIM at CT3 on day 3 in Pdfr han and Pdf 01 mutant LN v s than in control LN v s (Student's t test, both p,0.0001).At CT15, TIM levels are significantly reduced in Pdf 01 mutants compared to Pdfr han or control LN v s (Student's t test, both p,0.0003).(C) PDP1 immunostaining was quantified in LN v s of Control (blue) and Pdfr han mutant (red) larval brains on days 2 and 3 in DD.PDP1 oscillates in Pdfr han LN v s (ANOVA, F 3,37 = 46.22,p,0.0001).PDP1 levels were significantly higher at CT3 on day 3 in Pdfr han mutant LN v s than in control LN v s (Student's t test, p,0.01).(D and E) Histograms show the percentage of LN v clusters in which TIM (D) or PDP1 (E) was detected in either none or all four LN v s (''synchronized,'' green bars) or in one, two, or three LN v s (''desynchronized,'' red bars).(F and G) To further quantify desynchrony, the standard deviation (ST DEV) in TIM (F) or PDP1 (G) levels within a cluster of control, Pdf 01 , and Pdfr han mutant LN v s is shown as a box plot.Statistical comparisons by ANOVA with Tukey's post hoc test reveal significant increases in ST DEV in TIM in Pdf 01 (F 3,55 = 26.71,p,0.0001) and Pdfr han (F 3,53 = 12.13, p,0.0001) mutant LN v s compared to control LN v s at CT3 but not CT9.The ST DEV in PDP1 in Pdfr han mutant LN v s was also significantly elevated at CT3 but not CT9 (F 3,52 = 5.03, p = 0.004).The box shows the 25th-75th percentile, and whiskers represent the 95% confidence interval.doi:10.1371/journal.pbio.1001959.g001circadian rhythms in larval light avoidance [9].Larval DN 1 s also respond directly to PDF (Figure S2A).
We therefore used cry-Gal4 and Pdf-Gal80 (DN 1 .) to target transgene expression exclusively to DN 1 s [9].We first tested whether DN 1 ablation affected LN v synchrony by expressing Diptheria Toxin in DN 1 s (DN 1 .Dti).We found that TIM rhythms persisted in LN v s after DN 1 ablation (Figure S3D), indicating that LN v s do not require DN 1 s for oscillations per se.However, TIM levels at CT3 on both days 2 and 3 in DD were elevated in DN 1 -ablated larvae (Figure S3D).Examining TIM staining in DN 1 -ablated brains in DD revealed that 50% of LN v clusters were desynchronized at CT3 on days 2 and 3 in DD, a significant increase compared to controls (Figures 2C,D and S3A and Table S1).We observed similar increases in desynchrony of PDP1 expression when DN 1 s were ablated (Figures S3A,C,E and S6C-D and Table S1) with significantly higher levels at CT3 on day 3.We did not observe desynchrony in LD cycles (Figure 2D) or at CT9, just like Pdf 01 and Pdfr han mutants.We conclude that PDF signaling (Figure 1) and DN 1 s (Figure 2) normally maintain larval LN v molecular clock synchrony in constant darkness.

DN 1 Glutamate Synchronizes LN v s
To test this model further, we sought to identify the DN 1 signal and the relevant receptor in LN v s.Because larval DN 1 s are glutamatergic [32], we tested whether reducing DN 1 glutamate levels alters LN v molecular clock synchrony.Glutamate decarboxylase 1 (Gad1) was mis-expressed in DN 1 s, to convert glutamate into GABA [9,33], which cannot be released as DN 1 s do not produce the vesicular GABA transporter.Thus misexpression of Gad1 reduces presynaptic glutamate.This manipulation does not affect DN 1 viability, and their molecular clocks still oscillate [9].We found that overall TIM oscillations were relatively normal in DN 1 .Gad1 LN v s (Figure S4A).However, TIM levels were significantly elevated at CT3 in DN 1 .Gad1 larvae (Figure S4A).This is because DN 1 .Gad1 significantly increased LN v desynchrony, determined by comparing the standard deviation in TIM and PDP1 expression with control LN v s (Figures 3A,C,D and S4C and Table S1).Therefore, we conclude that DN 1 s release glutamate to synchronize LN v molecular clocks.

LN v s Perceive the Synchronizing Glutamate Signal Via mGluRA
Larval LN v s express two glutamate receptors: a metabotropic glutamate receptor (mGluRA, [32]) and a glutamate-gated Chloride channel (GluCl, [9]).To determine whether one of these receptors transduces the glutamate signal to synchronize LN v s, we used RNAi transgenes previously shown to reduce expression of mGluRA or GluCl [9,32].We found that reducing GluCl expression in LN v s had no effect on TIM and PDP1 oscillations or LN v synchrony (Figures 3B-D and S4B-C).In contrast, expressing mGluRA RNAi in LN v s produced similar molecular phenotypes to DN 1 ablation, with elevated TIM levels at CT3 and 75% of LN v s desynchronized (Figure 3B-D and Table S1).
As an independent way to manipulate mGluRA expression, we measured TIM levels at CT3 in LN v s of mGluRA 112b null mutant larvae (Figures 3B-C and S4D and Table S1).We found desynchronized LN v s in homozygous mGluRA 112b mutant larvae but not in heterozygous controls.We saw similar levels of desynchronization when measuring PDP1 levels in Pdf.mGluRA RNAi and mGluRA 112b mutant LN v s (Figures 3C and S4C-D).Taking these data together with our manipulations of DN 1 glutamate levels, we conclude that glutamate released by DN 1 s helps synchronize LN v oscillations via mGluRA.
We previously showed that LN v s require GluCl rather than mGluRA for circadian rhythms in the rapid light avoidance of larvae [9].Thus, a single neurotransmitter, glutamate, released by DN 1 s has two distinct functions depending on the receptor in LN v s that perceives the signal.Presumably the rapid action of the ionotropic receptor on LN v excitability [9] is best suited to regulate light avoidance behavior, whereas mGluRA acts on a slower timescale to regulate clock oscillations.

PdfR and mGluRA Cooperate to Maintain LN v Synchrony and Promote Strong TIM Oscillations
LN v s require two different signals to maintain synchrony, as reducing expression of either Pdfr or mGluRA desynchronized LN v molecular clocks.However, we only observed an increase in desynchronized LN v clusters at CT3 in Pdf.Pdfr RNAi or Pdf.mGluRA RNAi larval brains compared to controls, with most LN v clusters remaining synchronized at CT21.This suggested that the second signal-glutamate in Pdf.Pdfr RNAi and PDF in Pdf.mGluRA RNAi larvae-maintains some degree of LN v synchrony and we hypothesized that simultaneously reducing Pdfr and mGluRA expression would more strongly affect LN v clock synchrony.
We measured TIM and PDP1 oscillations in LN v s expressing transgenes targeting both Pdfr and mGluRA expression (Pdf.Pdfr RNAi +mGluRA RNAi ).We found that 88% of LN v clusters showed desynchrony in TIM protein levels at CT3 (Figures 4A-B and S5B and Table S1) and 75% for PDP1 (Figure S5A,C and Table S1).Pdf.Pdfr RNAi +mGluRA RNAi larvae also had significantly more desynchronized LN v clusters at CT21 and CT3 than control larvae (Figure S5A).Thus simultaneously reducing expression of both receptors dramatically increased the percentage of desynchronized LN v s compared to reducing Pdfr or mGluRA expression alone, indicating that PDF and glutamate signals work together to promote synchrony.
Although we observed a few individual LN v s with high TIM levels in Pdf.Pdfr RNAi +mGluRA RNAi larvae, overall TIM oscillations were almost completely lost (Figure 4C).This contrasts with the robust TIM oscillations of Pdf.Pdfr RNAi and Pdf.mGluRA RNAi single knock-down larvae (Figure S5E).Highamplitude TIM protein oscillations in LN v s thus depend on external signals, including PDF and glutamate, and are not fully cell-autonomous.Although PDP1 showed elevated desynchrony in Pdf.Pdfr RNAi +mGluRA RNAi LN v s (Figure S5A,C), overall PDP1 oscillations were relatively unaffected (Figure S5D).Thus Pdf.Pdfr RNAi +mGluRA RNAi LN v s are still partly functional.These data suggest that TIM is a more direct target than PDP1 in LN v s for the signaling pathways that transduce glutamate and PDF signals.
Do the reduced amplitude TIM rhythms in Pdf.Pdfr RNAi + mGluRA RNAi double mutant larvae affect behavioral rhythms?We had previously found that light avoidance rhythms require glutamate release from DN 1 s and transduction via GluCl in LN v s [9].Because TIM oscillations in LN v s remained intact in Pdf.GluCl RNAi larval brains (Figure S4B), we concluded that glutamate received by GluCl modulates LN v outputs rather than LN v molecular clocks [9].Knocking down either mGluRA or Pdfr individually in LN v s does not block TIM or PDP1 protein oscillations (Figure S5E-F) and larval light avoidance is still rhythmic, with peak levels at dawn (Figure 4D and [9]).However, we found that larvae with mGluRA and Pdfr expression simultaneously reduced in LN v s lose light avoidance rhythms (Figure 4D).This result suggests that TIM oscillations in LN v s are essential for light avoidance rhythms and that PDP1 rhythms alone cannot support larval rhythms.Overall, these data indicate the importance of extracellular signals for LN v s to oscillate normally and promote rhythmic behavior.

mGluRA and PdfR Are Activated at Different Times of Day in LN v s
Adult s-LN v s are most excitable at dawn [34,35] and drive the morning peak of locomotor activity [6,7].We previously showed that the same is likely true for the larval LN v s that control the dawn peak in light avoidance, whereas larval DN 1 s most likely signal at dusk [9].To test whether DN 1 s signal at dawn or dusk to promote LN v synchrony, we used a temperature-sensitive Shibire transgene (UAS-Shi ts [36]) to temporally block synaptic transmission.
Shi ts was expressed specifically in DN 1 s (DN 1 .Shi ts ), and larvae were maintained at the permissive temperature of 25uC for 4 days in LD and 1 day in DD.On the second day in DD, the temperature was elevated to the nonpermissive temperature of 31uC for 6 hours from either CT9 to CT15 (''CT12 shift'') or CT21 to CT3 (''CT24 shift'') to block DN 1 signaling around dusk or dawn, respectively (Figure 5A).Larval brains were dissected at CT3 on day 3 of DD (i.e., 12 hours after the end of a CT12 temperature shift or immediately after the end of a CT24 temperature shift).We found that 57% of LN v clusters showed desynchronized TIM levels when DN 1 synaptic transmission was blocked at dusk (DN 1 .Shi ts , 31uC at CT12) compared to 7% of control LN v s (UAS-Shi ts /+; Figure 5A-C and Table S1).Similarly, 36% of LN v s in DN 1 .Shi ts larvae shifted to 31uC at CT12 had desynchronized PDP1 levels compared to 0% of control LN v s (Figure S6A-B and Table S1).In contrast, blocking synaptic transmission from DN 1 s around dawn had no effect on LN v  S1).We therefore conclude that DN 1 signaling around dusk is required to synchronize LN v s.
To further test the idea that PDF and glutamate promote synchrony at different times of day, we took advantage of the synchronizing effect of LD cycles on Pdf 01 and DN 1 .Dti LN v s (Figures 2D, S1C, and S3B and Table S1).Based on the likely timing of LN v and DN 1 signals, wild-type LN v clocks should have received the PDF signal at subjective dawn by CT3 on day 1 in DD, but not yet received the glutamatergic signal at subjective dusk.Thus we predicted that Pdf 01 mutants would show desynchrony at this time point, whereas DN 1 .Dti LN v s, which still receive the PDF signal, would not.
We measured TIM and PDP1 levels in LN v s at CT3 on the first day of DD and found higher TIM and PDP1 levels and an increase in the variability of clock protein levels between LN v s in the same cluster in Pdf 01 mutants, indicating that LN v s are already desynchronized just 3 hours into DD (Figures 5D-E and S6C-D).In contrast, the LN v clocks in larvae with DN 1 s ablated remained synchronized at CT3 on the first day in DD, and desynchrony was first detected on day 2 (Figures 5D-E and S6C-D).
We interpret these data to mean that desynchrony in DN 1ablated larvae requires larvae to traverse subjective dusk when the DN 1 signal is released.Because desynchrony appears on different days in Pdf 01 and DN 1 -ablated larvae, this supports the model where LN v synchrony depends on PDF received at dawn and glutamate received at dusk.This is consistent with the previously reported timing of LN v excitability [34,35] and of the larval LN v and DN 1 signals that regulate light avoidance [9].

LN v cAMP Rhythms Require mGluRA and PdfR
PdfR and mGluRA are both G-protein coupled receptors.PdfR signals via Gas [18,19,21,37] and mGluRA can also alter cAMP levels [38].Because cAMP is a clock component in mammals [23] and likely also in flies [39,40], regulation of LN v cAMP levels by extracellular signals could maintain LN v synchrony and promote robust TIM oscillations.
We used the FRET-based Epac1-camps sensor [19] to measure basal cAMP levels on day 2 in DD.We first assayed control LN v s, focusing on their axonal termini near DN 1 projections [9].We found that cAMP levels, measured by the ratio of CFP/YFP, were highest at CT24, indicating that cAMP levels normally oscillate in LN v projections (Figure 6A).Strikingly, cAMP (CFP/YFP) oscillations were lost in the projections of both Pdf.Pdfr RNAi and Pdf.mGluRA RNAi larval LN v s (Figure 6A).
We noticed that Pdf.mGluRA RNAi LN v cAMP levels were significantly higher than controls at dusk (CT12), when DN 1 s signal for synchrony.This is consistent with data showing that mGluRA reduces cAMP levels by signaling via Gai [38], thereby opposing PdfR activity [18,37].To test this idea, we measured the responsiveness of LN v s to PDF with reduced mGluRA activity.We first generated a PDF response curve to determine the minimal PDF concentration that elicits an Epac1-camps response (Figure S7A-C).We then tested whether expressing mGluRA RNAi in Pdf.Epac1-camps LN v s altered this response (Figure 6B-C) using GluCl RNAi as a control.We found that mGluRA RNAi , but not GluCl RNAi , significantly increased LN v responsiveness to PDF (Figure 6C).Therefore, we propose that mGluRA acts in an opposite manner to PdfR and reduces intracellular cAMP.
To test if cAMP links to synchronized clock protein oscillations, we built on the recent identification of Adenylate cyclase 3 (AC3) as the specific Adenylate cyclase downstream of PdfR in LN v s [21].We tested whether AC3 is required for LN v synchronization by reducing expression of AC3 using two independent RNAi lines (Pdf.AC3 TRiP RNAi and Pdf.AC3 Vienna RNAi ) that reduce PDF responses in LN v s [21].We found that expressing each RNAi line in LN v s desynchronized TIM expression in 35%-40% of LN v clusters and PDP1 expression in 28%-35% of LN v clusters (Figure S8A-B and Table S1).Reducing AC3 expression in LN v s also significantly increased desynchrony measured by standard deviation in TIM and PDP1 expression (Figure S8C-D).
We conclude that PdfR and mGluRA regulate LN v cAMP levels at different times of day, presumably by regulating AC3 activity.This leads to a model in which LN v cAMP rhythms are generated by extracellular signals, with PDF/PdfR increasing cAMP via AC3 around dawn, whereas glutamate inhibits the response of LN v s to PDF via mGluRA by inhibiting AC3 around dusk (Figure 6D).cAMP oscillations then feed into the molecular clock, affecting TIM oscillations through an unknown mechanism, which will be a topic of future research.

PdfR and mGluRA Promote Molecular Clock Synchrony in Adult s-LN v s
We next tested whether our findings from larvae held true for the more complicated adult circadian system.Because we observed the most dramatic effects on larval LN v synchrony by simultaneously reducing Pdfr and mGluRA in LN v s, we measured the synchrony of s-LN v molecular clocks in Pdf.Pdfr RNAi + mGluRA RNAi adult flies.We found that many more Pdf.Pdfr RNAi +mGluRA RNAi s-LN v clusters were desynchronized than control s-LN v s (Figure 7A-C and Table S1), with extensive desynchrony detected at CT15 and CT21 on day 2 in DD and CT3 on day 3. TIM oscillations within Pdf.Pdfr RNAi +mGluR-A RNAi s-LN v s also displayed a reduced amplitude compared to control s-LN v s, although the effect was less pronounced than in larvae (Figure 7D).We also observed significant desynchrony at CT3 when either Pdfr or mGluRA expression was reduced in LN v s (Figure S9A).We conclude that PDF and glutamate (+/UAS-Gad1) and DN 1 .Gad1 experimental larvae.Genotypes in (B) are control (Pdf.+) and experimental larvae in which GluCl (Pdf.GluCl RNAi ) or mGluRA (Pdf.mGluRARNAi ) levels are reduced in LN v s, and mGluRA 112b /+ heterozygous control or mGluRA 112b mutant LN v s. (C) Histograms showing percentage of synchronized (green) or desynchronized (red) LN v clusters for TIM (left panel) or PDP1 (right panel) at CT3. Top: 14% of control (+/UAS-Gad1) LN v clusters are desynchronized compared to 71% of DN 1 .Gad1 LN v clusters by TIM staining, and 21% of control (+/UAS-Gad1) LN v clusters have detectable PDP1 expression compared to 64% in DN 1 .Gad1 brains.Middle: ,20% of Pdf.GluCl RNAi or +/UAS-mGluRA RNAi larval brains have desynchronized TIM levels compared to 62% of Pdf.mGluRA RNAi brains.Less than 20% of Pdf.GluCl RNAi or +/UAS-mGluRA RNAi larval brains have detectable PDP1 expression, compared to 71% of Pdf.mGluRA RNAi brains.Bottom: 50% of mGluRA 112b mutant LN v s show desynchronized TIM expression, compared to 8% of mGluRA 112b /+ controls.For PDP1, 29% of LN v clusters are desynchronized in mGluRA 112b mutants, compared to 4% of mGluRA 112b /+ controls.In addition, 3/24 mGluRA 112b mutants had all four LN v s expressing PDP1 compared to 0/25 control LN v clusters.(D) Box plots showing quantification of desynchrony by measuring ST DEV in TIM levels within a cluster in larval LN v s in control, DN 1 .Gad1, Pdf.GluCl RNAi , and Pdf.mGluRA RNAi larvae at CT3 on day 3 in DD.DN 1 .Gad1 (Student's t test, p = 0.0004) and Pdf.mGluRA RNAi (ANOVA with Tukey's post hoc test, F 2,50 = 5.597, p = 0.0064) significantly increase the ST DEV in TIM levels, reflecting increased LN v desynchrony, whereas Pdf.GluCl RNAi does not (ANOVA with Tukey's post hoc test, F 2,39 = 0.93, p = 0.40).doi:10.1371/journal.pbio.1001959.g003

Synchronizing Inputs to s-LN v s Regulate the Onset of Sleep
We next tested whether Pdf.Pdfr RNAi +mGluRA RNAi flies displayed behavioral defects.We compared the locomotor activity of Pdf.Pdfr RNAi +mGluRA RNAi flies to parental flies and to Pdf.GluCl RNAi flies to control for nonspecific effects of RNAi in LN v s, as GluCl RNAi does not affect larval LN v synchrony (Figure 3).Because Pdf.Pdfr RNAi +mGluRA RNAi flies have ,24 h locomotor activity rhythms in DD, we conclude that s-LN v desynchrony does not affect period length (Figure 8A and Table S2).However, we noticed that the activity of Pdf.Pdfr RNAi +mGluRA RNAi flies was much less consolidated than control or Pdf.GluCl RNAi flies, with bursts of activity visible in the subjective night when control flies are inactive (Figure 8A).
We calculated the average locomotor activity on the first 5 days in DD for each genotype.Pdf.Pdfr RNAi , Pdf.mGluRA RNAi , and Pdf.Pdfr RNAi +mGluRA RNAi flies displayed elevated levels of activity towards the end of subjective day and the beginning of subjective night (,CT6-18) compared to control and Pdf.GluCl RNAi flies (Figure 8B).Thus altering PDF and/or glutamate inputs to LN v s increases nighttime activity.
To further quantify these differences in nighttime activity, we used standard measures of sleep.We found decreased overall sleep levels when mGluRA expression was reduced either alone or with Pdfr (Figure S9B).In contrast, reducing Pdfr expression alone had no significant effect on overall levels of sleep (Figure S9B).Thus, we conclude that glutamate signals to LN v s regulate sleep levels, whereas PDF signals between LN v s do not regulate sleep.
Next, we quantified the transition between wakefulness and sleep in the evening by measuring how quickly flies fell asleep after CT12 (sleep latency).To ensure that any effects on the timing of sleep onset did not result from subtle period length differences between genotypes (Table S2), we measured sleep latency only on day 1 in DD when the phase of locomotor activity between genotypes is minimally affected by small period differences.We found that Pdf.Pdfr RNAi +mGluRA RNAi flies showed a significant increase in sleep latency compared to all other genotypes (Figure 8C).Their average sleep latency of 213 min compared to 113 min for UAS-Pdfr RNAi +UAS-mGluRA RNAi /+ control flies exceeds the 30 min period length difference between these genotypes (Figure 8C and Table S2).We observed no significant effects when either mGluRA or Pdfr expression was reduced singly (Figure 8C).
Thus, we conclude that blocking PDF and glutamate inputs to LN v s increases evening activity and delays sleep onset timing.We did not observe a significant effect of reducing mGluRA or Pdfr expression on sleep latency under LD cycles (Figure S9C), consistent with LD cycles synchronizing larval LN v clock oscillations (Figures 2D, 5D-E, S1C, and S3C).Although increased LN v desynchrony may not cause the sleep latency defects observed, it is clear that normal Pdfr and mGluRA activity in LN v s is required for normal sleep in DD.However, it is possible that desynchrony and sleep latency defects are separate phenotypes resulting from abrogated intercellular communication between clock neurons.

Synchronizing Larval Pacemaker Neurons Requires Two Signals
Feedback is an essential component in the molecular clocks that drive circadian behavior in animals [22].Here we demonstrate the importance of feedback across the circadian neural network to synchronize individual clock neurons.We showed that larval LN v s require two signals that cooperate to synchronize their clocks: PDF released at dawn from LN v s themselves and glutamate released by DN 1 s at dusk.The PDF signal received by PdfR in DN 1 s presumably also sets the phase of the DN 1 clock (Figure S2D) [8] to correctly time glutamate release that is then perceived by mGluRA in LN v s.Thus a feedback loop seems to exist at the circuit level, maintaining synchronized LN v clocks in DD.
Our experiments also reveal that synchronization of larval pacemaker neurons is a very active process, as LN v clocks were desynchronized 3 hours into the first subjective morning if they miss the dawn PDF signal.Consistent with the dual-synchronizer model, we see increased desynchrony when mGluRA and Pdfr expression is simultaneously reduced in LN v s (Figures 4B and S5A-C and Table S1).

Dual Roles for Glutamate in the Circadian Circuit
A DN 1 glutamate signal released around dusk is required for circadian rhythms of light avoidance when received by the ionotropic glutamate receptor GluCl in LN v s [9].We now show that DN 1 glutamate also promotes LN v synchrony when received by the metabotropic glutamate receptor mGluRA in LN v s.Thus a single neurotransmitter plays two distinct roles in the Drosophila circadian circuit depending on the receptor that receives the signal in LN v s: a rapid behavioral response to light mediated via GluCl and longer-term regulation of the 24 hour molecular clock via mGluRA.
Although mGluRA is not required for light avoidance [9], we found that larvae with reduced expression of both Pdfr and mGluRA lose larval light avoidance rhythms.This is consistent with the loss of strong TIM protein oscillations in the LN v s of Pdf.Pdfr RNAi +mGluRA RNAi larvae.These defects in the LN v molecular clock probably alter the timing of signals from LN v s and/or the phases of other clock neurons within the circuit.This contrasts with the role of GluCl, where glutamate received by GluCl directly regulates light avoidance by inhibiting the response of LN v s to ACh, independent of the LN v molecular clock [9].

Desynchronized Adult LN v s and Sleep
Synchronization of adult s-LN v s also depends on signaling via PdfR and mGluRA as .50% of s-LN v clusters were desynchronized at three of the four timepoints measured when expression of both Pdfr and mGluRA was reduced in LN v s.However, TIM oscillations in adult s-LN v s were not as severely impaired as in larval LN v s.The increased complexity of the adult clock neural circuit probably adds signals from neurons not present in larvae that promote synchronized and robust clock protein oscillations in adult s-LN v s.
Because the molecular clock in adult Pdf.mGluRA RNAi + Pdfr RNAi flies still oscillates, it is not surprising that locomotor activity is also still largely rhythmic.However, the desynchrony the number of larvae on the dark side of a Petri dish after 15 min.Light avoidance was assayed on day 2 (CT12, 18,24) or day 3 (CT6) of DD after prior LD entrainment.Control (Pdf.+)larvae (grey) and Pdf.Pdfr RNAi larvae (blue) show similarly phased light avoidance rhythms, peaking at dawn (twoway ANOVA, no Genotype6Time interaction, F 3,22 = 0.31, p = 0.82).Pdf.mGluRA RNAi +Pdfr RNAi larvae lose light avoidance rhythms (ANOVA F = 0.13, p = 0.94).doi:10.1371/journal.pbio.1001959.g004and reduced amplitude of TIM oscillations in Pdf.mGluRA RNAi + Pdfr RNAi LN v s correlates with increased nighttime activity and sleep latency.Desynchrony and increased activity could be independent consequences of reduced glutamate and PDF receptivity in LN v s.An alternative possibility is that because the molecular clock regulates daily firing rhythms of clock neurons [34,41,42], individual LN v s remain active at the wrong time of day in a desynchronized LN v cluster, preventing sleep.Indeed, if LN v s are electrically coupled like SCN neurons [43], then firing of a single LN v may cause the remaining LN v s in that cluster to fire earlier and/or later than programmed by their molecular clock.In addition, mistimed LN v signals in Pdf.Pdfr RNAi +mGluRA RNAi flies will affect the phases of other clock neurons in the circuit, which could also disrupt sleep timing.

Autonomy of the Molecular Clock
The loss of strong TIM protein oscillations in Pdf.Pdfr RNAi +mGluRA RNAi larval LN v s is surprising, as molecular clock oscillations in pacemaker neurons are often regarded as cellautonomous.Our data extend conclusions from the SCN showing that the VIP receptor, VPAC2R, is required for synchronized molecular clocks [14].By removing a second receptor simultaneously and by restricting our analyses to a defined subset of pacemaker neurons, we demonstrate that specifically blocking We observed a much stronger effect on TIM than on PDP1 oscillations in Pdf.mGluRA RNAi +Pdfr RNAi larval LN v s.It may be that TIM oscillations are relatively easily modified, allowing information from outside the cell to be integrated into the molecular clock, whereas a more robust PDP1 oscillation prevents LN v s overreacting to external stimuli.It is well-documented that TIM can be regulated at the posttranslational level in addition to transcriptional control [22], whereas PDP1 protein levels closely follow Pdp1 RNA levels [44].We propose that external signals mediated via PdfR and mGluRA mainly regulate the clock posttranslationally, and this is supported by recent findings [45][46][47].Testing this idea will require developing a combination of transcriptional and translational reporter genes.

The Role of cAMP in Maintaining Clock Neuron Synchrony
VIP and VPAC2R synchronize the mammalian SCN in a cAMP/Ca 2+ -dependent manner [14,16,23].VPAC2R is expressed more broadly than VIP, and some SCN neurons express both VIP and VPAC2R [48].This is highly reminiscent of   Drosophila, where Pdfr is found in both PDF+ and PDF-clock neurons [29].Because VIP/VPAC2R and PDF/PdfR are functionally similar and because both mediate synchronization of pacemaker neurons, discoveries about the roles of PDF/PdfR in Drosophila should be relevant to understand how synchrony is maintained across circadian neural circuits in general.In both flies and mammals, a reciprocal relationship between synchrony and clock protein amplitude seems to allow pacemaker neurons to be more precise and robust timekeepers than individual neurons.
Our data reveal a remarkable degree of conservation of clock circuit properties between mammals and Drosophila, echoing the conserved molecular basis of the circadian clock.The mechanisms promoting LN v synchrony in flies mirror the signaling pathways that make the SCN a more robust oscillator than other mammalian clock cells (reviewed in [4]).VIP and PDF are both required to synchronize the molecular clocks in different neurons, both promote robust oscillations of clock proteins within clock neurons, and they both likely signal through Gas and Adenylate cyclase [4].We have not yet determined the signaling pathways downstream of cAMP that link to clock protein oscillations, but they likely include PKA and/or Epac, which affect circadian rhythms in flies and mammals [23,49,50].Recent data show that PKA lies downstream of PDF and cAMP in Drosophila clock neurons (see Figure 9).In addition, Epac can regulate MAP kinase signaling, which is interesting because MAP kinase has also been proposed to lie downstream of PDF [51].
Our data provide evidence that the external signals that drive cAMP oscillations are received at different times of day.In the SCN, the amplitude of the cAMP rhythm is amplified by increased VIP signaling at dawn.cAMP levels decrease at dusk via falling VIP release and a release of the inhibition of Gai/o by RGS16 [5].However, the behavioral phenotypes of Rgs16 2/2 mice are modest, suggesting that additional signaling mechanisms operate.Based on the similarity of the mammalian and Drosophila systems, we predict that a second signal released around dusk is also required for normal SCN function.Two possible signals are GABA [52] and glutamate perceived via its metabotropic receptor [53].
In Drosophila, different clock neuron groups respond to specific environmental inputs such as light or temperature [10,11].Thus the true function of the cAMP oscillator in flies and mammals may be to integrate information from diverse clock neurons into the molecular clocks of all clock neurons, generating a single time of day for an animal.

Immunocytochemistry
All immunocytochemistry was carried out as in [44].We used the following antibodies: rat aTIM (from Amita Sehgal), rabbit aPDP1 [44], mouse aPDF [65], and rabbit aGFP (Sigma, St. Louis, MO).Images were scanned on a Leica SP2, SP6, or SP8 confocal microscope, with the same microscope used for a single experiment.The beginning and end of TIM staining was used to establish the limits of confocal stacks.The mean staining intensity for each channel for each neuron in every confocal stack was quantified using FIJI (http://pacific.mpi-cbg.de/wiki/index.php/Main_Page),with background levels of staining for each channel subtracted to control for variation in staining between brains.For each time course, the mean staining intensities for all LN v s in each brain lobe were averaged to give a single value for an LN v cluster.The average staining intensities per brain were then averaged to generate the time courses shown.
We used two methods to measure LN v synchrony.In a simple binary method, we used a cutoff of 20 arbitrary units (au) above background to determine if a cell produced TIM or PDP1 or not.We chose 20 au as it is the lowest number where protein levels are convincingly visible above background.An LN v cluster was then scored as ''desynchronized'' if they contained a mixture of LN v s with and without detectable TIM or PDP1, or ''synchronized'' if all four LN v s were the same.To more precisely quantify desynchrony, we also calculated the standard deviation in TIM or PDP1 mean staining intensities between individual LN v s within a single LN v cluster, producing a standard deviation in TIM or PDP1 staining intensity to use as a proxy for the level of desynchrony, allowing statistical comparisons of the data.

Behavioral Assays
Larval light avoidance assays were carried out as in [9].For adult locomotor activity experiments, adults were entrained to 12:12 LD cycles at 25uC for at least 3 days before transfer to DD. Locomotor activity was recorded using the DAM system (TriKinetics, Waltham, MA).

cAMP Measurements
Basal levels of Epac1-camps FRET were used to measure cAMP levels.Larval brains were dissected and mounted in hemolymphlike saline.To minimize the time from dissection to imaging (,1 hour), different genotypes were removed from DD, dissected, and scanned in the same order.LN v projections were imaged for CFP (460-490 nm) and YFP (528-603 nm) on an SP5 Leica confocal using a TD 458/514/594 dichroic 636 lens and 36 digital zoom at 100 Hz and 102461024 resolution, after excitation with a 458 nm laser.CFP and YFP levels were quantified using the Leica software.Background measurements of CFP and YFP (green, n = 26).Experimental genotypes are shown in red.Top left: Pdf.GluCl RNAi (n = 37).Top right: Pdf.mGluRA RNAi (n = 54).Bottom left: Pdf.Pdfr RNAi (n = 33).Bottom right: Pdf.Pdfr RNAi +mGluRA RNAi (n = 37).Activity between ,CT6 and 18 is elevated in Pdf.mGluRA RNAi , Pdf.Pdfr RNAi , and Pdf.Pdfr RNAi +mGluRA RNAi flies compared to controls or Pdf.GluCl RNAi .(C) Histogram shows the average sleep latency on the first day in DD.Pdf.Pdfr RNAi +mGluRA RNAi flies show significantly increased sleep latency compared to Pdf.+, +/UAS-mGluRA RNAI ; +/UAS-Pdfr RNAi , and Pdf.GluCl RNAi controls (ANOVA F = 6.83, p = 0.0003).doi:10.1371/journal.pbio.1001959.g008 were subtracted from raw CFP and YFP measurements and an average CFP to YFP ratio calculated for each image.For LN v projections, five boutons in each image were quantified for CFP and YFP as above and averaged to give a value per projection.
Live cAMP imaging was performed on larval LN v s as described in [66].Briefly, larval brains were dissected in hemolymph-like saline and mounted to the bottom of a 35-mm Falcon culture dish lid (Becton Dickenson Labware, Franklin Lakes, NJ), fitted with a Petri Dish Insert (PDI, Bioscience Tools, San Diego, CA).Brains were allowed to settle for 5-10 min to reduce movement during imaging.Images were acquired on an Olympus FV1000 laserscanning microscope (Olympus, Center Valley, PA) through a 606 (1.1N/A W, FUMFL N) Objective (Olympus, Center Valley, PA) using Fluoview software (Olympus).The Epac1-camps FRET sensor was imaged by scanning frames at 1 Hz with a 440-nm laser.An SDM510 dichroic mirror was used to separate CFP and YFP emission.Regions of interest were drawn around single LN v cell bodies in Fluoview.Peptides were bath applied using a micropipette after 30 s of baseline imaging.PDF was dissolved in 0.01% DMSO and vehicle controls consisted of 0.01% DMSO delivered at the same volume as peptide applications (45 mL bath application into 405 mL hemolymph-like saline).The lowest PDF dose that evoked a consistent response (100 nM) was used to assay differential responses of LN v s in which PDF or glutamate receptors had been knocked down in Figure 6.PDF was used at 100 mM in Figure S2.For each assay, no less than five larvae were imaged.Only one hemisphere was imaged per brain, and 1-4 LN v were imaged per brain.Processing and analysis of Epac1-camps data was as described [67].

Sleep Analysis
Fly locomotor activity was recorded in 5 min bins, using the DAM system (TriKinetics).Data analysis was performed using custom-written scripts in IgorPro (Wavemetrix).Sleep was defined as periods of immobility .5 min.Sleep latency was calculated for each fly on each day as the time from CT12 until the first sleep  [18][19][20][21].In this study, we show that glutamate (glu) signals received via mGluRA reduce cAMP levels, likely by inhibiting AC3.Differentially timed release of PDF and glutamate signals results in cAMP rhythms.PKA responds to cAMP to increase stability of the PER/TIM dimer via PER [46] and likely also via TIM (data here and inferred from non-LN v s [45]).Right panel: In non-LN v clock neurons, PDF signals via PDFR through Ga and unknown Adenyl cyclase(s) (AC) to boost intracellular cAMP.By analogy with what we show here for LN v s, we propose that an inhibitory signal released at a different time of day from PDF inhibits AC activity to generate a cAMP rhythm in non-LN v s.PKA responds to cAMP to increase stability of the PER/TIM dimer through TIM [45] and likely also PER (by analogy with LN v s [46]).doi:10.1371/journal.pbio.1001959.g009bout.Locomotor activity was calculated as the average number of beam crossings per 30 min bins and averaged for each genotype over the first 5 days in DD.Histograms showing the percentage of LN v clusters synchronized (green) or desynchronized (red) for TIM or PDP1 expression at CT3 in (left top panel) Pdf.+; +/UAS-Pdfr RNAi ; Pdf.Pdfr RNAi , (left bottom panel) tim .+;UAS-Pdfr RNAI /Pdf-Gal80; tim-Gal4.Pdfr RNAi , Pdf-Gal80, and (right) DN 1 .+;UAS-Dti/+; and DN 1 ablated larvae (DN 1 .Dti).(B and C) Box plots showing the distribution of ST DEV in PDP1 expression, with whiskers representing 95% confidence interval.(B) Pdf.Pdfr RNAi significantly increase ST DEV in PDP1 levels within an LN v cluster compared to both parental controls (ANOVA F 2,49 = 7.809, p = 0.0011), reflecting increased desynchrony.By ANOVA with Tukey's post hoc test, tim-Gal4; Pdf-Gal80.Pdfr RNAi is significantly different only from tim.+control LN v s (F 2,51 = 4.434, p = 0.017).However, by Student's t test, levels of PDP1 are also significantly increased in tim-Gal4; Pdf-Gal80.Pdfr RNA compared to UAS-Pdfr RNAi /Pdf-Gal80 controls (p = 0.046).(C) ST DEV in PDP1 levels between LN v s in each brain lobe at ZT3 and ZT9 in LD and CT3 and CT9 on day 3 in DD.Statistical comparisons by ANOVA with Tukey's post hoc test show a significant increase in ST DEV in PDP1 expression in DN 1 .Dti larvae compared to controls at CT3 only (F 2,49 = 8.59, p = 0.0006).(D) TIM and (E) PDP1 immunostaining was quantified for LN v s of Control (+/UAS-Dti; blue) and DN 1 -ablated (DN 1 .Dti, red) larval brains in ZT and days 2 and 3 in DD.DN 1 s are not required for LN v s to oscillate in DD (TIM, ANOVA, F 3,42 = 12.66, p,0.0001, and PDP1, ANOVA, F 3,28 = 23.71,p,0.0001).However, TIM levels were significantly higher at CT3 on days 2 and 3 in DN 1 .Dti larvae compared to controls (Student's t test, p = 0.0004 and p,0.0001, respectively), and PDP1 levels were significantly higher at CT3 on day 3 in DN 1 .Dti larvae compared to controls (Student's t test, p = 0.022).(TIF) mGluRA RNAi +Pdfr RNAi (green) LN v s.PDP1 oscillates relatively normally in Pdf.mGluRA RNAi +Pdfr RNAi larval LN v s (two-way ANOVA, no significant genotype effect, F 1,82 = 0.15, p = 0.6970).Average TIM (E) and PDP1 (F) levels are shown for Pdf.Pdfr RNAi (red) and Pdf.mGluRA RNAi (green) LN v s in DD on days 2 and 3. Pdf.mGluRA RNAi and Pdf.Pdfr RNAi larval LN v s display similar TIM and PDP1 oscillations.TIM, two-way ANOVA, no significant genotype effect (F 1,80 = 0.24, p = 0.6224) but a significant time effect (F 3,80 = 19.98,p,0.0001).For PDP1, no significant genotype effect (F 1,79 = 1.15, p = 0.2876) but a significant time effect (F 3,79 = 13.87,p,0.0001).(TIF)

Figure 1 .
Figure 1.Synchronized TIM and PDP1 oscillations in LN v s depend on PDF signaling.Larval LN v s were immunostained using TIM, PDP1, and PDF antibodies at CT 9, 15, 21, and 3 on days 2-3 in DD after 4 days prior entrainment to 12:12 LD cycles.Desynchrony data were calculated from 3-5 independent experiments, each with at least three brains.Error bars represent SEM.For total number of LN v clusters analyzed, see TableS1.

Figure 2 .
Figure 2. LN v and non-LN v clock neurons maintain LN v synchrony.All experimental lines and Pdf.+control larvae in RNAi experiments include UAS-Dcr-2, but this is omitted from written genotypes for simplicity.Desynchrony data were calculated from 3-4 independent experiments, each consisting of at least three but usually five or more brains.Total number of LN v clusters analyzed are in Table S1.** p,0.01; *** p,0.001.(A) Representative images of LN v s in control larvae (+/UAS-Pdfr RNAi ) or in larvae with reduced Pdfr levels in LN v s (Pdf.Pdfr RNAi ) or all clock neurons except LN v s (tim-Gal4; Pdf-Gal80.Pdfr RNAi ) immunostained for PDF (green), TIM (red), and PDP1 (blue) at CT3 on day 3 in DD.The lower panels for each genotype are the same images with the green channel (PDF) removed and replaced by a dashed white line outlining LN v s. (B) Box plots showing the ST DEV in TIM expression as in Figure 1.Statistical comparisons by ANOVA with Tukey's post hoc test show both Pdf.Pdfr RNAi (F 2,49 = 12.33, p, 0.0001) and tim-Gal4; Pdf-Gal80.Pdfr RNAi (F 2,51 = 8.158, p = 0.0008) significantly increase the ST DEV of TIM levels compared to parental controls, reflecting increased desynchrony.(C) Representative images of larval LN v s stained for PDF (green), TIM (red), and PDP1 (blue) at CT3 on day 3 in DD.From left to right, Control DN 1 .+,and +/UAS-Dti LN v clusters, and a representative desynchronized DN 1 .Dti LN v cluster.The green channel (PDF) has been removed from the lower panel and replaced by a dashed white outline of LN v s. (D) Box plots showing quantification of desynchrony through measurement of ST DEV in TIM expression in larval LN v s in control or DN 1 ablated larvae at ZT3, CT3, and CT9.DN 1 .Dti increases ST DEV at CT 3 compared to both parental controls (ANOVA with Tukey's post hoc test, F 2,49 = 10.5, p,0.0001).There was no significant difference between DN 1 .Dti and controls at ZT3 (Student's t test, p = 0.35) or CT9 (Student's t test, p = 0.31).doi:10.1371/journal.pbio.1001959.g002

Figure 3 .
Figure 3.A DN 1 glutamate signal mediated via mGluRA synchronizes LN molecular oscillations.All experimental lines and Pdf.+control larvae in RNAi experiments include UAS-Dcr-2, but this is omitted from written genotypes for simplicity.Desynchrony data were calculated from 2-5 independent experiments, each consisting of at least four brains.Total numbers of LN v clusters analyzed are in Table S1.* p,0.05; *** p,0.001.(A and B) Representative images of larval LN v s stained for PDF (green), TIM (red), and PDP1 (blue) at CT3 on day 3 in DD.Genotypes in (A) are control

Figure 4 .
Figure 4. PdfR and mGluRA promote high-amplitude TIM oscillations and larval behavioral rhythms.All experimental lines and Pdf.+control larvae also include UAS-Dcr-2 for RNAi experiments, but this is omitted from written genotypes for simplicity.Desynchrony data were calculated from 2-4 independent experiments, each consisting of at least five brains.Total number of LN v clusters analyzed are in Table S1.Error bars represent SEM.(A) Representative images of larval LN v s at CT 9, 15, 21, and 3 on days 2-3 in DD for control (+/UAS-mGluRA RNAI ; +/UAS-Pdfr RNAi ) or Pdf.mGluRA RNAi +Pdfr RNAi larval LN v s immunostained for TIM (red), PDP1 (blue), and PDF (green).PDF staining is removed from lower panels, with LN v s indicated by a white line.(B) Histogram showing the number of synchronized (green) or desynchronized (red) LN v clusters in control (+/UAS-mGluRA RNAI ; +/UAS-Pdfr RNAi ) or Pdf.mGluRA RNAi +Pdfr RNAi larval brains, determined by TIM staining at CT3. (C) Average TIM levels of control (blue) and Pdf.mGluRA RNAi +Pdfr RNAi (green) LN v s.TIM oscillations are dampened in Pdf.mGluRA RNAi +Pdfr RNAi larval LN v s (two-way ANOVA significant Genotype effect, F 1,102 = 119.53,p,0.0001, and Genotype6Time interaction, F 3,102 = 100.11,p,0.0001).(D) Larval light avoidance was measured by counting

Figure 5 .
Figure 5. PDF and glutamate signal at different times of day to regulate LN v cAMP levels.Statistical comparisons are by ANOVA with Tukey's post hoc test, unless otherwise stated.Desynchrony data were calculated from three independent experiments, each consisting of at least three brains.Total number of LN v clusters analyzed are in Table S1.Error bars show SEM.Whiskers represent 95% confidence.* p,0.05; ** p,0.01; *** p,0.005.(A-C) Larvae were subjected to a heat pulse (6 hours at 31uC) from either CT9 to CT15 on day 2 (CT12 shift) or from CT21 on day 2 to CT3 on day 3 of DD (CT24 shift).Larvae were then dissected at CT3 on day 3 of DD and immunostained with aTIM (red), aPDP1 (blue), and aPDF (green).(A) Representative images of control (+/UAS-Shi ts ) LN v s or LN v s of larvae expressing the temperature-sensitive allele of Shibire in DN 1 s (DN 1 .Shi ts ).At 31uC, Shi ts is inactive, blocking synaptic transmission.Left: Effect of heat pulse at CT12.Right: Effect of heat pulse at CT24/0.(B) Histograms showing the percentage of LN v clusters where TIM was detected in either none or all four of the four LN v s (''synchronized,'' green bars) or in one, two, or three LN v s (desynchronized, red bars).(C) Desynchrony was quantified as in Figure 1 by measuring ST DEV in TIM expression.A CT12 heat pulse significantly increased ST DEV of TIM expression in DN 1 .Shi ts brains compared to controls and to DN 1 .Shi ts larval brains with a CT24 heat pulse (F 3,60 = 6.423, p = 0.0008).(D) Larval LN v s were immunostained for TIM at ZT3 and CT3 on days 1 and 2 of DD in Control (+/UAS-Dti), DN 1 .Dti, and Pdf 01 mutants.DN 1 ablation and Pdf 01 mutants do not affect LN v TIM levels at ZT3 (F 3,41 = 1.53, p = 0.22).On the first day of DD, only Pdf 01 increases TIM expression in LN v s (F 3,51 = 11.43,p,0.0001).DN 1 .Dti increases TIM levels in LN v s on day 2 in DD (Student's t test, p = 0.0004).(E) Desynchrony of LN v s in LD and on days 1 and 2 of DD was quantified by measuring ST DEV of TIM expression in Control (+/UAS-Dti), DN 1 .Dti, and Pdf 01 mutants.The STDEV in TIM is significantly higher in Pdf 01 LN v s compared to control or DN 1 .Dti LN v s on the first day of DD, reflecting increased desynchrony (F 2,38 = 16.48,p,0.0001).DN 1 .Dti increases desynchrony as measured by TIM ST DEV only on day 2 in DD (Student's t test, p = 0.019).doi:10.1371/journal.pbio.1001959.g005

Figure 6 .
Figure 6.mGluRA and PdfR regulate intracellular cAMP.Statistical comparisons are by ANOVA with Tukey's post hoc test.Error bars show SEM.Whiskers represent 95% confidence.* p,0.05; ** p,0.01; *** p,0.001; **** p,0.0001.(A) Larvae were dissected and analyzed on day 2 in DD.CFP and YFP levels were measured in the projections of Pdf.Epac1-camps LN v s.The ratio of CFP/YFP reflects the basal level of cAMP.The CFP/YFP ratio oscillates in control (Pdf.Epac1-camps) LN v projections, peaking at CT24 (ANOVA F 3,62 = 2.933, p = 0.04).There is no significant oscillation in Pdf.Epac1-camps+mGluRA RNAi (F 3,59 = 0.815, p = 0.49) or Pdf.Epac1-camps+Pdfr RNAi (F 3,47 = 1.068, p = 0.37).The CFP/YFP ratio is significantly increased at CT12 in Pdf.Epac1-camps+mGluRA RNAi compared to control LN v s (F 2,38 = 5.021, p = 0.0017) but not in Pdf.Epac1-camps+Pdfr RNAi , consistent with glutamate signals inhibiting cAMP at CT12. (B) Averaged Epac-1-camps CFP/YFP ratio responses to bath application of 100 nM PDF or vehicle (arrow).The wild-type (Pdf.Epac1-camps) response to 100 nM PDF is shown in blue, and the wild-type response to vehicle is shown in black.Knockdown of GluCl (Pdf.Epac1-camps+GluCl RNAi , green) had no significant effect on the response to PDF, but knockdown of mGluRA (Epac1-camps+ mGluRA RNAi , magenta) significantly increased the cAMP response of LN v s to PDF.Vehicle traces represent 10 LN v cell bodies from five brains (10, 5), wild-type PDF (10, 5), Pdf.GluCl RNAi PDF (20, 9), and Pdf.mGluRA RNAi PDF (27, 12).(C) Comparison of mean maximum Epac-1-camps CFP/YFP ratio changes between 0 and 240 s [dashed line in (B)] [genotypes and sample sizes as in (B)].Application of 100 nM PDF significantly increased cAMP in LN v s of Pdf.Epac1-camps flies compared to vehicle (p,0.0001 by unpaired t tests).PDF responses of Pdf.Epac1-camps+GluCl RNAi LN v s were not significantly different from wild-type LN v s (p = 0.6217).PDF responses of Pdf.Epac1-camps+mGluRA RNAi LN v s were significantly higher than wild-type (p = 0.024) and Pdf.Epac1-camps+GluCl RNAi (p = 0.0193) LN v s. (D) Model: We propose that LN v s signal to each other via PDF around dawn.This signal is received by PdfR, which acts via Gas/AC3 to increase intracellular cAMP.DN 1 s release glutamate around dusk.This signal is received by mGluRA in LN v s, which acts via Gai to inhibit AC3 and reduce intracellular cAMP.Daily regulation of cAMP by external signals promotes robust TIM oscillations and LN v synchrony.doi:10.1371/journal.pbio.1001959.g006

Figure 7 .
Figure 7. mGluRA and PdfR help synchronize molecular oscillations in adult s-LN v s.Experimental lines include UAS-Dcr-2 for RNAi experiments, but this is omitted from written genotypes for simplicity.Desynchrony data were calculated from 2-3 independent experiments, each consisting of at least five brains.Total number of LN v clusters analyzed are in Table S1.Whiskers represent 95% confidence interval.* p,0.05.(A) Images of Control (+/UAS-mGluRA RNAI ; +/UAS-Pdfr RNAi , left) and Pdf.mGluRA RNAi +Pdfr RNAi (right) adult s-LN v s at CT9, 15, 21, and 3 on days 2-3 of DD immunostained for TIM and PDF.Examples for Pdf.mGluRA RNAi +Pdfr RNAi have been selected to show desynchronized LN v clusters, but synchronized LN v s were also observed at each time point.(B) Histogram showing the percentage of synchronized (green) or desynchronized (red) s-LN v s in each cluster assayed by TIM staining in control (left) or Pdf.mGluRA RNAi +Pdfr RNAi (right) brains at CT 9, 15, and 21 on day 2 and CT3 on day 3 of DD. (C) Box plots showing quantification of desynchrony through measurement of ST DEV in TIM expression in adult s-LN v s in control and Pdf.Pdfr RNAi + mGluRA RNAi flies at CT3 and CT9 on day 3 in DD.Pdf.Pdfr RNAi +mGluRA RNAi significantly increased desynchrony measured by ST DEV in TIM or PDP1 expression at CT3 but not at CT9 compared to controls (ANOVA with Tukey's post hoc test, F 3,36 = 5.313, p = 0.0039).(D) TIM expression in control (blue) or Pdf.mGluRA RNAi +Pdfr RNAi (red) s-LN v s.The amplitude of oscillation is dampened in Pdf.mGluRA RNAi +Pdfr RNAi compared to control LN v s (twoway ANOVA, genotype effect, F 1,82 = 9.77, p = 0.0025).Error bars show SEM.doi:10.1371/journal.pbio.1001959.g007

Figure 8 .
Figure 8. PdfR and mGluRA are required in LN v s for normal evening activity and timing of sleep onset.All experimental lines and Pdf.+control larvae also include UAS-Dcr-2 for RNAi experiments, but this is omitted from written genotypes for simplicity.Error bars show SEM.*** p, 0.001.(A) Locomotor activity was recorded for 3-4 days in LD cycles, followed by 10 days in DD (shaded area of actograms).Representative actograms are shown for Pdf.+ control flies and for Pdf.GluCl RNAi and Pdf.Pdfr RNAi +mGluRA RNAi experimental flies.(B) Graphs show average locomotor activity over the first 5 days in DD.Each panel shows two control genotypes: Pdf.+ (blue, n = 19) and +/UAS-mGluRA RNAI ; +/UAS-Pdfr RNAi

Figure 9 .
Figure 9. Model for regulation of cAMP levels and the molecular clock in clock neurons.Black arrows and text show established pathways; grey arrows and text reflect pathways inferred but not yet demonstrated.Left panel: In LN v s, PDF signals via PDFR and Ga/AC3 to boost intracellular cAMP[18][19][20][21].In this study, we show that glutamate (glu) signals received via mGluRA reduce cAMP levels, likely by inhibiting AC3.Differentially timed release of PDF and glutamate signals results in cAMP rhythms.PKA responds to cAMP to increase stability of the PER/TIM dimer via PER[46] and likely also via TIM (data here and inferred from non-LN v s[45]).Right panel: In non-LN v clock neurons, PDF signals via PDFR through Ga and unknown Adenyl cyclase(s) (AC) to boost intracellular cAMP.By analogy with what we show here for LN v s, we propose that an inhibitory signal released at a different time of day from PDF inhibits AC activity to generate a cAMP rhythm in non-LN v s.PKA responds to cAMP to increase stability of the PER/TIM dimer through TIM[45] and likely also PER (by analogy with LN v s[46]).doi:10.1371/journal.pbio.1001959.g009

Figure
FigureS1PDF signaling is required for LN v synchronization in DD. (A) Histograms showing the number of LN v s expressing TIM in each brain lobe in control, Pdf 01 , and Pdfr han larvae at CT3 and CT9.Because no TIM+ LN v s were detected in control brains at either time point, all LN v clusters were synchronized (green).TIM was detected in one, two, or three LN v s at CT3 in 50% of Pdf 01 mutant brains and in 58% of Pdfr han mutant brains; these are defined as desynchronized (red).No Pdf 01 or Pdfr han LN v s expressed TIM at CT9; thus all LN v clusters were synchronized.(B) Histograms showing the number of LN v s expressing PDP1 in each brain lobe in control and Pdfr han larvae at CT3 and CT9.No PDP1+ LN v s were detected in control brains at either time point; thus, all LN v clusters were synchronized (green).In Pdfr han larvae, PDP1 was detected in one, two, or three LN v s in 50% of brains examined at CT3; these are desychronized.No Pdfr han LN v s expressed PDP1 at CT9, and thus, all LN v clusters were synchronized.(C) Histograms showing the number of LN v s expressing TIM in each brain lobe in control, Pdfr han , and Pdf 01 larvae at ZT3. (TIF)Figure S2 Responses to PDF in DN1s and LNvs.Error bars represent SEM.* p,0.05; ** p,0.01; *** p,0.005.(A) Left: Average traces showing the responses of cry+ 39.Epac1-camps larval DN 1 s to 10 mM PDF (green), 10 mM PDF+2 mM TTX (purple), vehicle (blue), or vehicle+2 mM TTX (black).Shaded area around each line shows SEM.Brains were incubated in TTX for 20 min prior to the PDF application.Right: Histogram shows the maximum percentage change of CFP/YFP after bath application of Vehicle (Veh) or 10 mM PDF peptide 62 mM TTX. DN 1 s respond to PDF more strongly than to vehicle both without TTX (p = 0.0033) or with TTX (p = 0.0055).The p values were calculated using a multiple t test with Tukey's analysis.(B) Pdfr-Gal4 GMR18F07 was used to express UAS-GFP (green).Larvae were dissected at ZT21 and stained with PDF (blue) and PDP1 (red).This Pdfr enhancer-Gal4 localizes to DN1s but not DN 2 s. (C) Left: Average traces showing the responses of control (Pdf.Epac1-camps, blue), RNAi control (Pdf.baboRNAi ; UAS-Epac1camps, green), or Pdfr RNAi (Pdf.Pdfr RNAi +Epac1-camps, red) LN v s to application of 10 mM PDF.Average response of LN v s to application of vehicle is shown in black.Shaded area around each line shows SEM.Right: Histogram shows the maximum percentage change of CFP/YFP after bath application of 10 mM PDF peptide.Expression of Pdfr RNAi (Pdf.Pdfr RNAi +Epac1-camps) significantly reduces the maximum percentage change of CFP/YFP upon PDF application compared to control LN v s.Controls were sensor only (Pdf.UAS-Epac1-camps; p = 0.0045) and a line expressing a control RNAi (Pdf.baboRNAi UAS-Epac1-camps; p = 0.0426).The p values were calculated using the Mann-Whitney nonparametric t test.(D) DN 1 TIM oscillations on days 2 and 3 in DD show an altered phase in Pdfr han mutants compared to controls (two-way ANOVA, significant interaction between genotype and time, F 3,192 = 3.2, p = 0.03).(TIF) Figure S3 LN v and non-LN v clock neurons maintain LN v synchrony.For all RNAi experiments, Gal4/+ control and experimental lines include UAS-Dcr-2.Error bars represent SEM.* p,0.05; ** p,0.01; *** p,0.001; **** p,0.0001.(A)

Figure
Figure S6 Dawn PDF and Dusk glutamate signals alter LN v PDP1 expression.All statistical comparisons are by ANOVA with Tukey's post hoc test unless otherwise stated.Error bars represent SEM.Whiskers represent 95% confidence interval.* p,0.05; ** p,0.01; *** p,0.005.(A) Histograms showing the percentage of LN v clusters showing synchronized/desynchronized PDP1 expression in control or DN 1 .shits LN v s after a 6 hour 31uC heat pulse centered at CT12 or CT24.(B) Box plots representing the ST DEV of PDP1 expression in LN v s of control or DN 1 .shits larvae dissected at CT3 on day 3 of DD after a 31uC heat pulse centered at CT12 or CT24 on day 2 of DD.A heat pulse at CT12 significantly increased the ST DEV in PDP1 expression of DN 1 .Shi ts larval LN v clusters (Student's t test, CT12 versus 24, p,0.01), but did not affect controls.(C) Larval LN v s were immunostained for PDP1 at ZT3 and at CT3 on days 1 and 2 of DD in Control (+/UAS-Dti), DN 1 .Dti, and Pdf 01 mutants.DN 1 ablation or the Pdf 01 mutation do not affect LN v PDP1 levels at ZT 3 (F 2,34 = 1.70, p = 0.2).Pdf 01 increases TIM expression in LN v s on the first day of DD, whereas DN 1 .Dti does not (F 2,38 = 8.62, p = 0.0008).(D) Desynchrony of LN v s in ZT and on the first and second days of DD was quantified by measuring ST DEV of PDP1 expression in Con (+/UAS-Dti), DN 1 .Dti, and Pdf 01 mutants.There is no difference between genotypes at ZT3 (F 2,34 = 2.89, p = 0.07).ST DEV in PDP1 is significantly higher in Pdf 01 LN v s compared to control or DN 1 .Dti LN v s on the first day of DD, reflecting increased desynchrony (F 2,38 = 4.62, p = 0.016).DN 1 .Dti increases desynchrony as measured by PDP1 ST DEV only on day 2 in DD (Student's t test, p = 0.041).(TIF) Figure S7 Dose response of larval LN v s to bath-applied PDF.Error bars show SEM.* p,0.05; ** p,0.01; *** p,0.001.(A) Averaged Epac-1-camps CFP/YFP ratio responses to bath application (triangle) of a range of PDF concentrations and vehicle.Sample sizes were as follows: vehicle, seven LN v cell bodies imaged from five brains (7, 5), PDF 10 28 M: (12, 5), PDF 10 27 M: (15, 6), PDF 3610 27 M: (17, 6), PDF 10 26 M: (13, 5), Table S1 Number of LN v s expressing TIM or PDP1 in each LN v cluster analyzed.Numbers indicate the number of clusters with zero, one, two, three, or four LN v s expressing TIM or PDP1 for each genotype.(PDF) Table S2 Behavioral periods and strengths of behavioral rhythms in adult flies with altered glutamate and PDF receptor expression in LN v s. (PDF)