A Series of Suppressive Signals within the Drosophila Circadian Neural Circuit Generates Sequential Daily Outputs

Abstract

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

Keywords: Drosophila; calcium; circadian physiology; modulation; neuropeptide.

Figure 1. PDF and cyclic light-dark conditions phase-delay the Ca²⁺ rhythm of E-pacemaker LNd

(A) Daily Ca²⁺ activity patterns of five major Drosophila circadian pacemaker groups in wild type (WT) flies under 12h light: 12h dark (LD) cycle (n = 6 flies). Left, average Ca²⁺ transients. Right, Ca²⁺ phase distribution: each colored dot represents calculated peak phase of one group in one fly; colored arrows are mean vectors for different groups; the arrow magnitude describes the Ca²⁺ phase coherence among different flies in a specific pacemaker group. Yellow aspect indicates 12 h period of light stimulation. (B) Daily Ca²⁺ activity patterns in WT flies under constant darkness (DD) conditions (n = 12 flies). Darker gray aspect indicates subjective night. (C) Daily Ca²⁺ activity patterns in pdf⁰¹ mutants under LD cycle (n = 5 flies). (D) Daily Ca²⁺ activity patterns in pdf⁰¹ mutants under DD (n = 11 flies). (E) Quantification of Ca²⁺ phase shifts described in panels (A–D). The mean phase of each group in WT controls under LD is set to zero (*p<0.01: Watson-Williams test). Error bars denote SEM. (F) Average locomotor activity of WT flies (n = 16 flies) in LD cycles (averaged across 6 days) and in the first day under DD (DD1). Dots indicate SEM. (G) Average locomotor activity of pdf⁰¹ mutants (n = 15 flies) in LD and DD1.

Figure 2. PDFR signaling regulates Ca²⁺ rhythms in M and E pacemakers by cell autonomous mechanisms

(A–D) Daily Ca²⁺ activity patterns of five major pacemaker groups under DD (A) in WT flies (n = 5 flies), (B) in pdfr mutants (n = 6 flies), (C) in an E cell rescue design - wherein E pacemakers (three out of six LNd and the 5^th s-LNv) express PDFR in the pdfr mutant background (n = 6 flies), and (D) in an M cell rescue – wherein flies with M pacemakers (four PDF-positive sLNv) express PDFR in the pdfr mutant background (n = 8 flies). Left, schematics of PDFR expression patterns in major pacemaker groups. Filled circles indicate all cells in the group express PDFR. Half circles indicate approximately half of cells in the group express PDFR. Middle, average Ca²⁺ transients. Right, Ca²⁺ phase distributions. (E) Quantification of Ca²⁺ phase shifts described in panels (A–D). The mean phase of each group in WT controls under DD is set to zero (*p<0.05, **p<0.01, and n.s. - not significant: Watson-Williams test). Error bars denote SEM. (F) Quantification of peak widths (the full width at half maximum) for Ca²⁺ transients in all groups and conditions (*p<0.05: Two-way ANOVA, followed by Bonferroni post hoc test).

Figure 3. Synthetic PDF application suppresses and/or delays Ca²⁺ activity in vivo

(A) Left, schematic illustrating yoked pairs of WT and pdfr mutant flies for pharmacological tests. Right, representative images of LNd and s-LNv pacemakers in such Drosophila pairs responding differentially to 10⁻⁰⁵M synthetic PDF. Axis above denotes the circadian time of recordings and the CT7 time point of synthetic PDF application. (B) Averaged Ca²⁺ transients of M pacemakers (s-LNv, orange) and E pacemakers (LNd, blue) responding to (top) PDF or (bottom) vehicle from (left) WT or (right) pdfr mutant flies. Gray traces represent individual cells in trials (PDF: n = 5 pairs; vehicle: n= 4 pairs). (C–D) Ca²⁺ signal changes by (C) PDF treatment or (D) vehicle treatment measured at the point that WT pacemakers displayed a maximal reduction in response to PDF (*p<0.01: single-sample t-test). The average increase in Ca²⁺ signals in WT LNd with vehicle treatment represents their normal daily Ca²⁺ peak that occurs during these recording periods (cf. Figure 1C). (E) Ca²⁺ activity responses in pdf⁰¹ mutants to application of vehicle saline at the time indicated by vertical arrows under DD (n = 6 flies). (F) Ca²⁺ activity responses in pdf⁰¹ mutants to single synthetic PDF application under DD (n = 5 flies). PDF was present in the initial saline bath as indicated by the vertical orange arrow at a peak concentration of 10⁻⁵ M. This initial time point represents the peak time of Ca²⁺ in s-LNv (CT0 – orange arrow). PDF was slowly washed out by perfusion (gray curve below the orange arrow denotes PDF concentration). (G) Ca²⁺ activity responses in pdf⁰¹ mutants to single synthetic PDF application at CT5 under DD (n = 6 flies). (H) Ca²⁺ activity responses to two serial applications of synthetic PDF in pdf⁰¹ mutants under DD (n = 6 flies). The first dose was at CT0 (as in panel F), and the second at CT5 (~ at the l-LNv Ca²⁺ peak time) both denoted by vertical orange arrows; constant perfusion followed each application. (I) Quantification of Ca²⁺ phase shifts produced by synthetic PDF; the mean phase of each group in pdf⁰¹ mutants under DD is set to zero (*p<0.05: Watson-Williams test).

Figure 4. PDFR signaling delays Ca²⁺ activation in diverse pacemakers

(A–E) Daily Ca²⁺ activity patterns of five major pacemaker groups under DD (A) in flies with all pacemakers over-expressing PDFR in pdfr mutants (n = 6 flies); (B) in flies with PDF-positive neurons (s-LNv and l-LNv) over-expressing PDFR in an otherwise WT background (n = 5 flies); (C) in flies with l-LNv over-expressing PDFR in pdfr mutants (n = 6 flies); (D) in flies with l-LNv alone over-expressing PDFR (by c929-gal4) in pdfr mutants (n = 4 flies); (D) in flies with all pacemakers except PDF-positive neurons over-expressing PDFR in pdfr mutants (n = 6 flies); and (E) in flies with E pacemakers (three out of six LNd and the 5^th s- LNv) expressing PDFR in pdfr mutants (n = 5 flies). Dashed arrows on the clock face indicate the mean phases of those groups in WT flies (cf. Figure 1A). (F) Quantification of l-LNv Ca²⁺ phase shifts described in panels (A–C). The mean phase of each group in WT controls under DD is set to zero (cf. Figure 1C; *p<0.05, ***p<0.001: Watson-Williams test). (G) Quantification of Ca²⁺ phase shifts in PDF-negative, PDFR-positive pacemakers described in panels (D–E). The mean phase of each group in WT controls under DD is set to zero (cf. Figure 1C; the colors of the asterisks correspond to the cognate pacemaker groups; *p<0.001: Watson-Williams test).

Figure 5. Light pulses phase-shift circadian pacemaker Ca²⁺ rhythms in vivo

(A) Averaged Ca²⁺ transients in the five circadian pacemaker groups in the three days following 15 min light pulses delivered either in the dead zone (CT9), or in the phase-delay zone (ZT17), or in the phase-advance zone (ZT21). Bars indicate the time of light pulses. Horizontal lines on top indicate separate 24 h imaging sessions for individual flies that were tiled to synthesize three-day patterns (CT9: n = 14 flies; ZT17: n = 18 flies; ZT21: n = 14 flies). (B) Ca²⁺ phase distributions of five circadian pacemaker groups in three circadian cycles immediately following the three different light pulse stimuli: CT9, ZT17, and ZT21. (C–E) Ca²⁺ phase response curves (PRC) plotted over the course of the three circadian cycles: (C) day one, (D) day two, and (E) day three. The Ca²⁺ phase shifts compared to Ca²⁺ phases in unstimulated flies (from Figure 1A) after three different light pulse stimuli. Phase-shifts that lacked coherence (p>0.05: Rayleigh test) were excluded.

Figure 6. sNPF is required for DN1 Ca²⁺ rhythms in vivo

(A) Daily Ca²⁺ activity patterns in tim > sNPF RNAi flies (sNPF knockdown) under DD conditions (n = 5 flies). Dashed arrows on the clock face indicate the mean phases of DN1 in WT flies (cf. Figure 1C). DN1 Ca²⁺ activity displayed poor phase coherence among flies (p>0.1: Rayleigh test). (B) Daily Ca²⁺ activity patterns in flies with sNPF knockdown in M pacemakers, pdf > sNPF RNAi under DD conditions (n = 5 flies). DN1 Ca²⁺ activity was rhythmic but phase-shifted compared to WT controls (p<0.01: Watson-Williams test). (C–D) Daily Ca²⁺ activity patterns under LD cycles in (C) tim > sNPF RNAi flies (n = 7 flies) and (C) pdf > sNPF RNAi flies (n = 6 flies). (E–F) Average locomotor activity in the first day under DD of (E) tim > sNPF RNAi flies (n = 15 flies) and (F) pdf > sNPF RNAi flies (n = 32 flies). Dots indicate SEM. See Figure 1F for a comparison to locomotor profiles in a control (WT) genotype. (G–H) Average locomotor activity in LD cycles (averaged across 6 days) of (G) tim > sNPF RNAi flies and (H) pdf > sNPF RNAi flies. See Figure 1F for a comparison to locomotor profiles in a control (WT) genotype.

Figure 7. sNPF suppresses DN1 Ca²⁺ activity

(A–D) As in Figure 3A–D, yoked pairs of WT flies entrained to different circadian time (PDF: n = 3 pairs; vehicle: n= 3 pairs). DN1 Ca²⁺ signals from flies in subjective night were reduced by 10⁻⁰⁵M synthetic sNPF application. The nighttime DN1 Ca²⁺ signals increased with vehicle treatment, reflecting their normal peak phase (cf. Figure 1C).

Figure 8. Model of PDF-, sNPF- and light-mediated interactions that in concert set sequential Ca2+ activity phases of the different pacemaker groups

(A) The position of each pacemaker group on the circle indicates its Ca²⁺ peak phase. Both PDF and sNPF signals suppress the receivers (LNd and DN3 for PDF; DN1 for sNPF) from being active when senders (s-LNv for PDF; s-LNv and LNd for sNPF) are active. Light cycles act together with PDF to delay LNd Ca²⁺ phases (Figure 1A&C). (B) Loss of neuropeptide-mediated interactions caused alterations in network Ca²⁺ activity patterns: pdf/pdfr deficient (Figure 1D and Figure 2B), M-cell sNPF knockdown (Figure 7B), E-cell sNPF knockdown (Figure S6F), and PDF cell ablation (Figure S7). (C) Ca²⁺ phase shifts occurring within 24 h following 15 min light pulses suggest that light also regulates PDF and sNPF signals (Figure 5).