Differentially timed extracellular signals synchronize pacemaker neuron clocks

Abstract

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

Figure 1. Synchronized TIM and PDP1 oscillations in LN_vs depend on PDF signaling.

Larval LN_vs 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 Table S1. * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001. (A) Representative images of y w (Control, top panels), Pdf⁰¹ 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_vs. Pdf⁰¹ LN_vs 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 Pdf⁰¹ (green) LN_vs on days 2 and 3 in DD. TIM oscillates in Pdfr^han (ANOVA F_3,37 = 13.68, p<0.0001) and Pdf⁰¹ (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 Pdf⁰¹ mutant LN_vs than in control LN_vs (Student's t test, both p<0.0001). At CT15, TIM levels are significantly reduced in Pdf⁰¹ mutants compared to Pdfr^han or control LN_vs (Student's t test, both p<0.0003). (C) PDP1 immunostaining was quantified in LN_vs of Control (blue) and Pdfr^han mutant (red) larval brains on days 2 and 3 in DD. PDP1 oscillates in Pdfr^han LN_vs (ANOVA, F_3,37 = 46.22, p<0.0001). PDP1 levels were significantly higher at CT3 on day 3 in Pdfr^han mutant LN_vs than in control LN_vs (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_vs (“synchronized,” green bars) or in one, two, or three LN_vs (“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, Pdf⁰¹, and Pdfr^han mutant LN_vs 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 Pdf⁰¹ (F_3,55 = 26.71, p<0.0001) and Pdfr^han (F_3,53 = 12.13, p<0.0001) mutant LN_vs compared to control LN_vs at CT3 but not CT9. The ST DEV in PDP1 in Pdfr^han mutant LN_vs 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.

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_vs in control larvae (+/UAS-Pdfr^RNAi) or in larvae with reduced Pdfr levels in LN_vs (Pdf>Pdfr^RNAi) or all clock neurons except LN_vs (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_vs. (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_vs stained for PDF (green), TIM (red), and PDP1 (blue) at CT3 on day 3 in DD. From left to right, Control DN₁>+, and +/UAS-Dti LN_v clusters, and a representative desynchronized DN₁>Dti LN_v cluster. The green channel (PDF) has been removed from the lower panel and replaced by a dashed white outline of LN_vs. (D) Box plots showing quantification of desynchrony through measurement of ST DEV in TIM expression in larval LN_vs in control or DN₁ ablated larvae at ZT3, CT3, and CT9. DN₁>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 DN₁>Dti and controls at ZT3 (Student's t test, p = 0.35) or CT9 (Student's t test, p = 0.31).

Figure 3. A DN₁ 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_vs stained for PDF (green), TIM (red), and PDP1 (blue) at CT3 on day 3 in DD. Genotypes in (A) are control(+/UAS-Gad1) and DN₁>Gad1 experimental larvae. Genotypes in (B) are control (Pdf>+) and experimental larvae in which GluCl (Pdf>GluCl^RNAi) or mGluRA (Pdf>mGluRA^RNAi) levels are reduced in LN_vs, and mGluRA^112b/+ heterozygous control or mGluRA^112b mutant LN_vs. (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 DN₁>Gad1 LN_v clusters by TIM staining, and 21% of control (+/UAS-Gad1) LN_v clusters have detectable PDP1 expression compared to 64% in DN₁>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_vs 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_vs 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_vs in control, DN₁>Gad1, Pdf>GluCl^RNAi, and Pdf>mGluRA^RNAi larvae at CT3 on day 3 in DD. DN₁>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).

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_vs 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_vs immunostained for TIM (red), PDP1 (blue), and PDF (green). PDF staining is removed from lower panels, with LN_vs 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_vs. TIM oscillations are dampened in Pdf>mGluRA^RNAi+Pdfr^RNAi larval LN_vs (two-way ANOVA significant Genotype effect, F_1,102 = 119.53, p<0.0001, and Genotype×Time interaction, F_3,102 = 100.11, p<0.0001). (D) Larval light avoidance was measured by counting 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 (two-way ANOVA, no Genotype×Time 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).

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 31°C) 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 αTIM (red), αPDP1 (blue), and αPDF (green). (A) Representative images of control (+/UAS-Shi^ts) LN_vs or LN_vs of larvae expressing the temperature-sensitive allele of Shibire in DN₁s (DN₁>Shi^ts). At 31°C, 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_vs (“synchronized,” green bars) or in one, two, or three LN_vs (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 DN₁>Shi^ts brains compared to controls and to DN₁>Shi^ts larval brains with a CT24 heat pulse (F_3,60 = 6.423, p = 0.0008). (D) Larval LN_vs were immunostained for TIM at ZT3 and CT3 on days 1 and 2 of DD in Control (+/UAS-Dti), DN₁>Dti, and Pdf⁰¹ mutants. DN₁ ablation and Pdf⁰¹ 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 Pdf⁰¹ increases TIM expression in LN_vs (F_3,51 = 11.43, p<0.0001). DN₁>Dti increases TIM levels in LN_vs on day 2 in DD (Student's t test, p = 0.0004). (E) Desynchrony of LN_vs in LD and on days 1 and 2 of DD was quantified by measuring ST DEV of TIM expression in Control (+/UAS-Dti), DN₁>Dti, and Pdf⁰¹ mutants. The STDEV in TIM is significantly higher in Pdf⁰¹ LN_vs compared to control or DN₁>Dti LN_vs on the first day of DD, reflecting increased desynchrony (F_2,38 = 16.48, p<0.0001). DN₁>Dti increases desynchrony as measured by TIM ST DEV only on day 2 in DD (Student's t test, p = 0.019).

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_vs. 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_vs (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_vs 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_vs of Pdf>Epac1-camps flies compared to vehicle (p<0.0001 by unpaired t tests). PDF responses of Pdf>Epac1-camps+GluCl^RNAi LN_vs were not significantly different from wild-type LN_vs (p = 0.6217). PDF responses of Pdf>Epac1-camps+mGluRA^RNAi LN_vs were significantly higher than wild-type (p = 0.024) and Pdf>Epac1-camps+GluCl^RNAi (p = 0.0193) LN_vs. (D) Model: We propose that LN_vs signal to each other via PDF around dawn. This signal is received by PdfR, which acts via Gαs/AC3 to increase intracellular cAMP. DN₁s release glutamate around dusk. This signal is received by mGluRA in LN_vs, which acts via Gαi to inhibit AC3 and reduce intracellular cAMP. Daily regulation of cAMP by external signals promotes robust TIM oscillations and LN_v synchrony.

Figure 7. mGluRA and PdfR help synchronize molecular oscillations in adult s-LN_vs.

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_vs 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_vs were also observed at each time point. (B) Histogram showing the percentage of synchronized (green) or desynchronized (red) s-LN_vs 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_vs 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_vs. The amplitude of oscillation is dampened in Pdf>mGluRA^RNAi+Pdfr^RNAi compared to control LN_vs (two-way ANOVA, genotype effect, F_1,82 = 9.77, p = 0.0025). Error bars show SEM.

Figure 8. PdfR and mGluRA are required in LN_vs 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 (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).

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_vs, PDF signals via PDFR and Gα/AC3 to boost intracellular cAMP –. 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 and likely also via TIM (data here and inferred from non-LN_vs [45]). Right panel: In non-LN_v clock neurons, PDF signals via PDFR through Gα and unknown Adenyl cyclase(s) (AC) to boost intracellular cAMP. By analogy with what we show here for LN_vs, 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_vs. PKA responds to cAMP to increase stability of the PER/TIM dimer through TIM and likely also PER (by analogy with LN_vs [46]).