DD circadian behavior parameters.

<p>See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001810#s4" target="_blank">Materials and Methods</a> for details of behavior quantification.</p>a<p>Only one rhythmic fly, therefore there is no SEM.</p><p>*<i>p</i><0.014 versus both parental controls.</p

PDF(+) sLNv make direct synaptic connections with DN1p neurons.

<p>(A) PDF-labeled sLNv terminals in the dorsal brain (red) are tightly intermingled with DN1p neurites marked by membrane-GFP expression using the <i>Clk4.1-G4</i> driver. (B) GRASP labeling (green) demonstrates direct cell-cell contact between the DN1p and anti-PDF labeled sLNv (red). (B) Reconstituted GFP label (green) is observed only at putative points of contact between the two cell groups. (C) Higher magnification of the region outlined in (B) reveals close apposition of the two labels (white arrowheads) but little overlap, delineating the synaptic architecture.</p

The PDF-cell clock does not impact PDP1ε in non-PDF clock neurons.

<p>Labels and nomenclature are the same as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001810#pbio-1001810-g004" target="_blank">Figure 4</a>. PDP1ε images are displayed in NIH ImageJ lookup table 5 Ramps (inverted) for visibility. Cell group (N): (A) LNd (88–125), (B) DN1 (175–281). *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001.</p

Loss of PDF reduces TIM levels in non-PDF circadian neurons.

<p>TIM staining in the <i>per⁰¹</i> mutant background was compared in the presence or absence of endogenous PDF. Brains were collected and fixed at CT24 of DD1. Images are displayed as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001810#pbio-1001810-g004" target="_blank">Figures 4</a>–<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001810#pbio-1001810-g006" target="_blank">6</a>. Bar graphs are normalized TIM staining intensity measurements combined from two independent experiments. Cell group (N): LNd (85–92), DN1 (112–135). ***<i>p</i><0.001.</p

PDF activates a TTX resistant cationic inward current in Drosophila DN1p neurons.

<p>(A) Representative voltage clamp recording at ZT6. Changes in ionic currents were measured from a ramp protocol form −100 mV to +100 mV in 300 ms. Black and blue traces represent currents recorded respectively before and after focal application of PDF 50 µM. (B) Crop from (A) showing inward currents from −100 mV to −60 mV. (C) Time course of PDF induced inward rectifying current measured at −100 mV without (black trace) or with TTX 10 µM (blue trace). Time course of FSK (D) or cAMP (E) induced inward rectifying current measured at −100 mV (for (E) black trace is without cAMP dialysis in the pipette, and red trace is when cAMP was added into the intracellular solution). (F) Histograms showing reduced inward current when Na⁺ was replaced from the extracellular solution with NMDG and comparable inward currents in control (CT), forskolin (FSK), or cAMP induced inward currents (respectively, 2.75±0.56 pA. pF⁻¹, <i>n</i> = 5 in control, 1.39±0.21 pA.pF⁻¹, <i>n</i> = 3 in low sodium, 2.27±0.44 pA.pF⁻¹, <i>n</i> = 3 with FSK and 3±0.83 pA.pF⁻¹, <i>n</i> = 2 in control).</p

Non-PDF (E-cell) pacemaker neuron specific modulation of PKA activity can phenocopy or rescue <i>pdfr</i>- behaviors.

<p>E-cells were targeted using <i>cry13-G4</i> and <i>pdf-G80</i>. <i>U-PKA-mC</i>* is a constitutively active PKA catalytic subunit that lacks the ability to bind to regulatory subunits. Graphs are as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001810#pbio-1001810-g001" target="_blank">Figure 1</a>. Genotype (N). (A) pdf-G80/+;cry13-G4/+ (15), (B) pdf-G80/+;cry13-G4/U-PKA-R1dn (10), (C) pdfr-;pdf-G80/+;cry13-G4/U-PKA-R1dn (26), (F) pdf-G80/U-PKA-mC*;cry13-G4/+ (15). (G) pdfr-;U-PKA-mC*/+ (22), (H) pdfr-;pdf-G80/U-PKA-mC*;cry13-G4/+ (48). (D,E,I,J) magnified overlays of morning (D,I) and evening (E,J) for the indicated color-coded genotypes as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001810#pbio-1001810-g001" target="_blank">Figure 1</a>. *<i>p</i><0.05 versus both G4 and UAS parental controls. a, <i>p</i><0.05 versus UAS parental control, <i>p</i> = 0.056 versus G4 parental control. b, <i>p</i><0.05 versus <i>pdfr</i>-;<i>U-PKA-mC</i>*/+, not significant versus <i>pdf-G80</i>/U<i>-PKA-mC</i>*;<i>cry13-G4</i>/+. Complete quantification appears in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001810#pbio-1001810-t001" target="_blank">Table 1</a>.</p

<i>cwo-G4</i>><i>U-PKA-R1dn</i> expression reduces TIM levels in the absence of PER in LD.

<p>PKA-R1dn was expressed broadly in the circadian system using <i>cwo-G4</i> and restricted from PDF cells using <i>pdf-G80</i>. Data are displayed as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001810#pbio-1001810-g004" target="_blank">Figure 4</a>. Cell group (N): (A) sLNv (36–90), (B) LNd (49–130), (C) DN1 (42–195). *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001.</p

Model for a bifurcation in the PDFR signaling pathway controlling the molecular clock and neuronal excitability.

<p>PDFR acts through Gs and adenylate cyclase (AC) to increase levels of cAMP which may directly activate a cyclic-nucleotide-gated (CNG) channel (green pathway) to acutely depolarize the cell and increase the action potential firing rate. cAMP also activates PKA, promoting TIM stability and progression of the molecular clock (blue pathway). Light activates CRY, which promotes TIM degradation. The molecular clock also controls PKA transcripts, thereby controlling signal transduction to the clock through a feedback mechanism.</p

PDF cell clocks specifically target TIM in LNds and DN1s in constant darkness.

<p>Single confocal slices showing PDF, PER, and TIM staining. <i>per⁰¹</i>;;<i>U-per</i>/+ (control, black lines) and <i>per⁰¹</i>;<i>pdf-G4</i>/+;<i>U-per</i>/+ (pdfPER, blue lines). sLNvs are marked with anti-PDF. TIM and PER images are displayed in NIH ImageJ lookup table 5 Ramps (inverted) for visibility. PDF images used to identify sLNv are in gray scale. Cells (N): (A) sLNv (53–87), (B) LNd (74–122), (C) DN1 (57–208). Average cell intensities were normalized to PPP CT6 = 1 before combining measurements from three (TIM) experiments. In some cases error bars (SEM) are very small and obscured by the data point. In no case were error bars omitted. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001.</p

PKA function in DN1p neurons.

<p>(A) Reduction of PKA activity in DN1p has no effect on the phase of evening activity, but reduces morning anticipation. Graphs are as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001810#pbio-1001810-g001" target="_blank">Figure 1</a>. Genotype (N). (A) Clk4.1-G4/+ (19), (B) Clk4.1-G4/U-PKA-R1dn (14). (C and D) are overlays of morning and evening activity as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001810#pbio-1001810-g001" target="_blank">Figure 1</a>. PKA subunits are transcriptionally regulated by the circadian clock in DN1ps. DN1ps were marked with <i>Clk4.1-G4</i>><i>U-GFP</i> in wt (black) or <i>per⁰¹</i> (blue) background, dissociated, sorted, and analyzed by quantitative RT-PCR for PKA subunit transcripts (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001810#s4" target="_blank">Materials and Methods</a>). (E) PKA-C1, (F) PKA-R1, (G) PKA-R2. Error bars are SEM.</p