Synchronous Drosophila circadian pacemakers display nonsynchronous Ca²⁺ rhythms in vivo

Abstract

In Drosophila, molecular clocks control circadian rhythmic behavior through a network of ~150 pacemaker neurons. To explain how the network's neuronal properties encode time, we performed brainwide calcium imaging of groups of pacemaker neurons in vivo for 24 hours. Pacemakers exhibited daily rhythmic changes in intracellular Ca(2+) that were entrained by environmental cues and timed by molecular clocks. However, these rhythms were not synchronous, as each group exhibited its own phase of activation. Ca(2+) rhythms displayed by pacemaker groups that were associated with the morning or evening locomotor activities occurred ~4 hours before their respective behaviors. Loss of the receptor for the neuropeptide PDF promoted synchrony of Ca(2+) waves. Thus, neuropeptide modulation is required to sequentially time outputs from a network of synchronous molecular pacemakers.

Fig. 1. Ca²⁺ activity patterns in circadian pacemaker neurons in vivo

(A) Schematic representations of bimodal behavioral rhythms (top) that are driven by a pacemaker network that display synchronous, unimodal molecular clocks. (B) Map of the eight major clock pacemaker groups in the fly brain. Numbers in parentheses indicate the cell number per group. (C) Schematic to illustrate method for long-term in vivo imaging; the head is immersed in saline while the body remains in an air-filled enclosure (see Methods for details). (D) A representative image of tim>GCaMP6s signals showing the locations of five identifiable pacemaker groups. (E) Representative images showing 24-h Ca²⁺ activity patterns of five identifiable groups. (F) Average Ca²⁺ transients in the five pacemaker groups as a function of Circadian Time (n=13 flies). Gray aspect indicates the period of lights-off during the preceding six days of 12hour:12hour photoentrainment. (G) Phase distributions of 24 hour Ca²⁺ transients in different pacemaker groups (data from G). Each colored dot outside of the clockface represents the calculated peak phase of one group in one fly as described in Methods. Colored arrows are mean vectors for the different clock neuron groups. The arrow magnitude describes the phase coherence of Ca²⁺ transients in a specific pacemaker group among different flies (n=13, not all 5 groups were visible in each fly due to the size of the cranial windows – see Table S1). Ψ_{M, E} is the phase difference between M cells (s-LNv) and E cells (LNd). (H) The average activity histogram of tim>GCaMP6s,mCherry.NLS flies in the first day under constant darkness (DD1). Arrows indicate behavioral peak phases (orange: morning, blue: evening). Dots indicate SEM (n=47 flies). (I) Phase distributions of behavioral peaks indicated by arrows in (I) (asterisks: peak phases of individual flies; orange: morning, blue: evening). Ψ_{M, E} is the phase difference between morning and evening behavioral peaks. (J) Comparing phase differences between M cells (s-LNv) and other pacemaker groups (potential E cells): the difference between s-LNv and LNd (Ψ_M,LNd) best compared to the behavioral Ψ_{M, E}. n.s. = not significant; “*” denotes significantly different groups (P <0.05) by ANOVA followed by post hoc Tukey tests. Ψ_M,LNd matched behavioral Ψ_{M, E} (t-test, p=0.91; f-test, p=0.65). Error bars denote SEM.

Fig. 2. Spontaneous Ca²⁺ activity patterns are CRY-independent and reflect pacemaker functions

(A) Schematic of PDFR-expressing clock neurons: neuronal groups and sub-groups driven by pdfr(B)-gal4 are filled and color-coded; those imaged for GCaMP6s signals are underlined. (B–F) As Fig. 1G–K: (B) Ca²⁺ transients in three PDFR+ clock neuron groups and subgroups (n=10 flies): activities in the three PDFR+ LNd and in the single 5^th s-LNv are similar (Pearson’s r=0.89). (C) Ca²⁺ rhythm phases from panel (B). (D) The DD1 locomotor activity of pdfr(B)>GCaMP6s,mCherry.NLS flies (n=8). (E) The phases of behavioral peaks from panel (D). (F) Phase differences from M cells (s-LNv) to both LNd and the 5^th s-LNv matched behavioral Ψ_{M, E} (ANOVA, p=0.7239).

Fig. 3. Effects of environmental information and molecular clocks on the spatiotemporal patterns of Ca²⁺ activity in the pacemaker network

(A and E) Ca²⁺ transients: (A) under long (16:8 LD) photoperiod (n=6 flies) and (E) under short (8:16 LD) photoperiod (n=6 flies). (B and F) Ca²⁺ rhythm phases (B) under long photoperiod and (F) under short photoperiod. The shaded circular sectors indicate the 8 hours (B) and 16 hours (F) lights-out periods. Note that M cells (s-LNv, orange) peaked around lights-on and E cells (LNd, blue) peaked before lights-off regardless of photoperiod. (C and G) The phases of behavioral peaks in DD1 after 6 days of photoperiodic entrainment: (C) long photoperiod (n=13 flies) and (G) short photoperiod (n=12 flies). (See Fig S5 for more details). (D and H) Ψ_M,LNd matches behavioral Ψ_{M, E} under long photoperiod (t-test, p=0.32; f-test, p=0.88) and under short photoperiod (t-test, p=0.30; f-test, p=0.16). (I) Arrhythmic Ca²⁺ transients in per⁰¹ mutants (n=5 flies). (J) Phase coherence of Ca²⁺ transients was poor among per⁰¹ flies. (K) Amplitude of Ca²⁺ transients (maximum dF/F) was significantly smaller in per⁰¹ and in per^S mutants (vs. control flies, Mann-Whitney test, *p<0.1, ***p<0.001). (L) Ca²⁺ transients in per^S mutants (n=6 flies). (M) Ca²⁺ rhythm phases of per^S mutants. (N) Phases of behavioral peaks corresponding to Ca²⁺ rhythm phases in panel (M) (n=16 flies). (O) Ψ_M,LNd matched behavioral Ψ_{M, E} (t-test, p=0.83; f-test, p=0.13).

Fig. 4. Requirement of PDFR signaling for staggered waves of Ca²⁺ transients among the pacemaker groups

(A) Ca²⁺ transients in five pacemaker groups in pdfr^han5304 mutants (n=7 flies). (B) Ca²⁺ rhythm phases from panel (A): LNd and DN3 were phase-shifted towards s-LNv. (C) Ca²⁺ transients in pdfr mutant flies that are restored by a large BAC-recombineered pdfr-myc transgene (Rescue 1, n=6 flies). (D) Ca²⁺ rhythm phases from panel (C). (E) The phase shifts in mutants were fully rescued by restoring PDFR (two-way ANOVA followed by a Bonferroni post-hoc test, *p<0.001). Colors in this panel indicate genotype. (F) Ca²⁺ transients in three pacemaker groups targeted by pdfr(B)-gal4 in pdfr^han5304 mutants (n=6 flies). (G) Ca²⁺ rhythm phases from (F). (H) Ca²⁺ transients in pdfr mutant flies that are restored by pdfr(B)-gal4>pdfr (Rescue 2, n=6 flies). (I) Ca²⁺ rhythm phases from (H): the PDFR+ LNd and the single 5^th s-LNv display restored phases, but lack strong phase coherence (Rayleigh test, p>0.1) (also see Fig. S12). (J) Phase shifts in mutant flies were partially restored by restoring pdfr in subsets of PDFR⁺ cells (two-way ANOVA followed by a Bonferroni post-hoc test, *p<0.001). Colors in this panel indicate genotype.