Synchronous Drosophila circadian pacemakers display nonsynchronous Ca2+ rhythms in vivo

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Science  26 Feb 2016:
Vol. 351, Issue 6276, pp. 976-981
DOI: 10.1126/science.aad3997

Layered versatility atop circadian clocks

The circadian clock evolved to allow cells or organisms to anticipate changes in physiological requirements associated with Earth's 24-hour light/dark cycle. Some activities, however, need to occur out of phase with the core clock. Liang et al. imaged changes in intracellular concentration of Ca2+ in populations of neurons in the fruit fly brain. Although the underlying clock was synchronous, the rhythms of Ca2+ changes corresponded with distinct timing of activities associated with activity of the particular neuronal populations. Proper coordination of these distinct phases required expression of the neuropeptide pigment-dispersing factor and its receptor.

Science, this issue p. 976


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 Ca2+ 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. Ca2+ 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 Ca2+ waves. Thus, neuropeptide modulation is required to sequentially time outputs from a network of synchronous molecular pacemakers.

Circadian clocks help animals adapt their physiology and behavior to local time. The clocks require a highly conserved set of genes and proteins (1) operating through molecular feedback loops to generate robust rhythms that produce a 24-hour timing signal (2). These clocks are expressed by pacemaker neurons, which themselves are assembled into an interactive network (3). Through network encoding and cellular interactions, pacemaker neurons in the suprachiasmatic nucleus (SCN) of the mammalian brain coordinate many circadian rhythmic outputs (47). To study how molecular clocks couple to network encoding and how network encoding relates to specific behavioral outputs, we conducted an in vivo brainwide analysis of the circadian pacemaker network in Drosophila across an entire 24-hour day.

This network contains ~150 synchronized pacemaker neurons (8, 9) (fig. S1), yet it produces biphasic behavioral outputs—the morning and evening peaks of locomotor activity (Fig. 1A). The molecular clocks are entrained by environmental cues and by network interactions, for example, by release of the neuropeptide PDF (pigment-dispersing factor) (10). Genetic mosaic studies indicate that morning and evening peaks of locomotor activity are controlled by distinct pacemaker groups (1114) (Fig. 1B). We reasoned that (i) synchronous signals from the pacemaker network might diverge in downstream circuits or (ii) the pacemaker network might itself generate different timing signals to downstream circuits. To explore this, we developed an in vivo imaging assay to monitor the intracellular Ca2+ concentration ([Ca2+]i) in pacemaker cell bodies over a ~24-hour period (Fig. 1C and supplementary materials). Intracellular Ca2+ dynamics directly reflect amounts of neuronal activity, and Ca2+ imaging allows monitoring activity across neuronal ensembles (15).

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 displays synchronous, unimodal molecular clocks. (B) Map of the eight major clock pacemaker groups in the fly brain; those imaged for GCaMP6s signals are underlined. Numbers in parentheses indicate the cell number per group. (C) Illustration of method for long-term in vivo imaging; the head is immersed in saline while the body remains in an air-filled enclosure (see supplementary materials for details). (D) A representative image of tim>GCaMP6s signals showing the locations of five identifiable pacemaker groups. (E) Representative images showing 24-hour Ca2+ activity patterns of five identifiable groups. Scale bars, 20 μm. (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 6 days of 12:12 photoentrainment. (G) Phase distributions of 24-hour Ca2+ transients in different pacemaker groups [data from (F)]. Each colored dot outside of the clock face represents the calculated peak phase of one group in one fly, as described in the supplement. 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 five groups were visible in each fly because of the size of the cranial windows; see table S1). ΨM,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 (H) (asterisks, peak phases of individual flies; orange, morning; blue, evening). ΨM,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 (ΨM,LNd) best compares to the behavioral ΨM,E. n.s., not significant; asterisk denotes significantly different groups (P < 0.05) by analysis of variance (ANOVA) followed by post hoc Tukey tests. ΨM,LNd matched behavioral ΨM,E (t test, P = 0.91; f test, P = 0.65). Error bars denote SEM.

We used objective-coupled planar illumination (OCPI) microscopy (16), which illuminates an entire focal plane simultaneously; this method accelerates volumetric imaging and reduces phototoxicity caused by repeated illumination. To permit imaging, we made cranial holes in the heads of living tim > GCaMP6s flies, which express the Ca2+ sensor GCaMP6s in all pacemaker neurons (15) (Fig. 1C), and monitored [Ca2+]i in five of the eight major pacemaker groups: small lateral neuron ventral (s-LNv), large lateral neuron ventral (l-LNv), lateral neuron dorsal (LNd), dorsal neuron 1 (DN1), and dorsal neuron 3 (DN3) (Fig. 1D). Each of the five groups displayed a prominent peak of [Ca2+]i during the 24-hour recording sessions, and each peak had distinct timing (Fig. 1E). To test whether these Ca2+ dynamics reflected intrinsic circadian patterning, we began 24-hour recording sessions at different zeitgeber times (ZT). In all such recordings, the peaks of Ca2+ activity reflected the pacemaker group identity, not the time at which recordings began (fig. S2). Thus, Ca2+ varies in pacemaker neurons systematically as a function of the time of day according to biologically defined rules of entrainment (Fig. 1F).

Three Drosophila pacemaker groups (l-LNv, s-LNv, and DN1p) show morning peaks of electrical activity when measured in acutely dissected brains (1719). Thus, the phases of Ca2+ rhythms we observed are roughly coincident with, or slightly anticipate, their peak electrical activity. Ca2+ rhythms produced by different pacemaker groups were similar in amplitude (Fig. 1F) but different in waveform (fig. S3) and phase (Fig. 1G). We confirmed our results using the fluorescence resonance energy transfer (FRET)–based cameleon2.1 imaging method (20), for which the ratio of fluorescence between yellow and cyan fluorescent proteins reflects [Ca2+]i independent of the abundance of the sensor. [Ca2+]i estimated by this assay exhibited a factor of ~2 circadian variation, with temporal patterns consistent with those obtained with GCaMP6s (fig. S4). In addition, the [Ca2+]i rhythms did not result from experimental activation of CRYPTOCHROME (fig. S5). These observations demonstrate that the Drosophila pacemaker network exhibits stereotyped and diverse spatiotemporal patterns of Ca2+ activity during the course of the 24-hour day.

We compared this diversity of Ca2+ activity patterns with the diversity of pacemaker functions. Pacemaker functions have been revealed by genetic mosaic experiments, as exemplified by the categorization of M (morning) and E (evening) cells (1114). These autonomous oscillators primarily drive the morning and evening peaks of locomotor activity, respectively. The phase relationships (ΨM,E) between the peaks of Ca2+ rhythms in canonical M (s-LNv) and E (LNd) cells and the two daily peaks of locomotor activity were highly correlated (Fig. 1, H to J). In M cells, the Ca2+ rhythm peaked toward the end of the subjective night, whereas in E cells it peaked toward the end of the subjective day (Fig. 1F). The ~10-hour phase difference between Ca2+ rhythms in M and E pacemakers is similar to the ~10-hour phase difference between the morning and evening behavioral peaks (Fig. 1J). Thus, M and E pacemaker Ca2+ activations precede by ~4 hours the behavioral outputs they control. The distinct phases of Ca2+ rhythms in the other three pacemaker groups (l-LNv, DN1, and DN3) may also involve the morning and evening behavioral peaks, or may regulate other, distinct circadian-gated outputs.

The E category of pacemakers includes the LNd as well as the fifth s-LNv (1114). However, the LNd is a heterogeneous group of neurons that exhibits diverse entrainment properties (21); likewise, the critical fifth s-LNv could not be unambiguously identified with tim-GAL4. To better understand the function of these subsets of E pacemakers, we used a PDF receptor [pdfr(B)] GAL4 driver (22); this driver restricts GCaMP6s expression to s-LNv, to three of six LNds, and to the single fifth s-LNv (Fig. 2A). The three PDFR-expressing LNds and the fifth s-LNv displayed the same basic E cell pattern of Ca2+ activity—a peak in late subjective day—which suggests that they both function as circadian pacemakers (Fig. 2B). Thus, the phase difference between Ca2+ rhythms in these PDFR-expressing M and E cell groups again matched that between the morning and evening behavioral activity peaks (Fig. 2, C to F).

Fig. 2 Ca2+ rhythms can be resolved within individual components of the E pacemaker groups.

(A) Schematic of PDFR-expressing clock neurons. Neuronal groups and subgroups driven by pdfr(B)-gal4 are filled and color-coded; those imaged for GCaMP6s signals are underlined. (B to F) As in Fig. 1, F to J: (B) Ca2+ transients in three PDFR+ clock neuron groups and subgroups (n = 10 flies). Activities in the three PDFR+ LNds and in the single fifth s-LNv are similar (Pearson’s r = 0.89). (C) Ca2+ rhythm phases from (B). (D) The DD1 locomotor activity of pdfr(B)>GCaMP6s,mCherry.NLS flies (n = 8). (E) The phases of behavioral peaks from (D). (F) Phase differences from M cells (s-LNv) to both LNd and the fifth s-LNv matched behavioral ΨM,E (ANOVA, P = 0.7239).

M and E cell categorization supports a classic model of seasonal adaptation (23) wherein a two-oscillator system responds differentially to light and so can track dawn and dusk independently. For example, under long-day conditions, light accelerates a “morning” clock and decelerates an “evening” clock. If these Ca2+ rhythms are critical output features of M and E cells, their properties may also reflect differences in photoperiodic entrainment. We entrained flies under either long-day (16 hours light, 8 hours dark) or short-day (8 hours light, 16 hours dark) conditions. In these flies, the phase difference between the morning and evening behavioral activity peaks tracked dawn and dusk (fig. S6). Likewise, the phases of pacemaker Ca2+ rhythms also tracked dawn and dusk (Fig. 3, A, B, E, and F, and fig. S7). Regardless of the photoperiodic schedule, the s-LNv (M cells) always peaked around dawn, whereas the LNd (E cells) always peaked before dusk (Fig. 3, B to D and F to H). Thus, Ca2+ activity patterns within the pacemaker network correspond to the circadian temporal landmarks of dawn and dusk.

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 under long photoperiod (B) and under short photoperiod (F). The shaded circular sectors indicate lights-out periods of 8 hours (B) and 16 hours (F). 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 details. (D and H) ΨM,LNd matches behavioral ΨM,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 (Rayleigh test, P > 0.5). (K) Amplitude of Ca2+ transients (maximum dF/F) was significantly smaller in per01 and in perS mutants (versus control flies; Mann-Whitney test, *P < 0.1, ***P < 0.001). (L) Ca2+ transients in perS mutants (n = 6 flies). (M) Ca2+ rhythm phases of perS mutants. (N) Phases of behavioral peaks corresponding to Ca2+ rhythm phases in (M) (n = 16 flies). (O) ΨM,LNd matched behavioral ΨM,E (t test, P = 0.83; f test, P = 0.13).

We tested whether changes in the molecular oscillator would alter the patterns of [Ca2+]i. We used different alleles of the gene period, which encodes a state variable of the Drosophila circadian clock. In per01 (null) mutant flies, which lack inherent rhythmicity in their molecular oscillators and in free-running behavior (24, 25) (fig. S8), all clock neurons showed reduced rhythmicity in [Ca2+]i. The amplitudes of Ca2+ fluctuations were reduced by half (Fig. 3, I and K) and coherence was lost within groups (Rayleigh test, P > 0.5; Fig. 3J and table S1). In fast-running perS mutant flies, which have a free-running period of ~19 hours (24, 25) (fig. S9), the Ca2+ rhythms were phase-shifted (Fig. 3, L and M, and fig. S10), consonant with the direction of behavioral phase shifts (Fig. 3N and fig. S9). The phase difference between Ca2+ rhythm peaks in perS M and E pacemakers still matched the phase difference between M and E behavioral peaks (Fig. 3, N and O). Thus, molecular clocks determine the pace of Ca2+ rhythms in the pacemaker network.

To explore how synchronous molecular clocks can have phases of Ca2+ activation that are staggered by many hours, we tested whether PDF, which mediates interactions between pacemakers, was required. Flies bearing the severely hypomorphic han5304 mutation of the PDF receptor show unimodal or arrhythmic behavior patterns under constant darkness (26) (fig. S11 and table S2). In these flies, we found that the Ca2+ rhythms in M cells (s-LNv and DN1) were unaffected, but they were phase-shifted in LNd and DN3, such that these two groups now produced Ca2+ rhythms around dawn, roughly in synchrony with M cells (Fig. 4, A and B). The phase of l-LNv did not change, consistent with the absence of PDF sensitivity by this pacemaker group (27). The phase shifts in LNd and DN3 were fully restored by the expression of complete pdfr from a bacterial artificial chromosome (BAC) transgene (Fig. 4, C to E, “rescue 1,” and fig. S11). Thus, PDF, which promotes synchronization of molecular clocks under constant conditions (10, 28), is also needed to properly stagger their Ca2+ activity phases across the day. Whether the phases of the l-LNv and DN1 are set by other intercellular signals remains to be determined.

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

(A) Ca2+ transients in five pacemaker groups in pdfrhan5304 mutants (n = 7 flies). (B) Ca2+ rhythm phases from (A): LNd and DN3 were phase-shifted toward 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 (C). (E) The phase shifts in mutants were fully rescued by restoring PDFR (two-way ANOVA followed by Bonferroni post hoc test, *P < 0.001). Colors indicate genotype. (F) Ca2+ transients in three pacemaker groups targeted by pdfr(B)-gal4 in pdfrhan5304 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 fifth s-LNv display restored phases but lack strong phase coherence (Rayleigh test, P > 0.1) (see also fig. S12). (J) Phase shifts in mutant flies were partially restored by restoring pdfr in subsets of PDFR+ cells (two-way ANOVA followed by Bonferroni post hoc test, *P < 0.001). Colors indicate genotype.

We further examined the pdfr mutant phenotype at higher cellular resolution [pdfr(B)> GCaMP6s; Fig. 2A]. The PDFR-expressing E cell groups (the three PDFR-expressing LNd and the fifth s-LNv) displayed phase shifts similar to those of the entire LNd group (Fig. 4, F and G). When pdfr expression was restored just in these subsets of pacemaker neurons (with GAL4-UAS), both behavior and Ca2+ rhythms were partially restored (Fig. 4, H to J, “rescue 2,” and fig. S11 and table S2). The phase of the fifth s-LNv was fully restored, which suggests that PDFR signaling is required for cell-autonomous setting of Ca2+ phase in this pacemaker group. However, in rescue 2, a single LNd typically remained active around dawn, whereas two LNds were active around dusk (fig. S12), which we interpret as a partial restoration or a nonautonomous phase-setting mechanism for LNd.

Our results show that molecular clocks drive circadian rhythms in the neural activity of pacemakers. Temporally patterned neural activity encodes different temporal landmarks of the day in a manner that reflects the different functions of the pacemaker groups. The homogeneous molecular clock produces sequential activity peaks by a mechanism dependent on PDFR signaling. By generating diverse phases of neural activity in different pacemaker groups, the circadian clock greatly expands its functional output.

Supplementary Materials

Materials and Methods

Supplementary Text

Figs. S1 to S12

Tables S1 and S2

References (2944)

References and Notes

Acknowledgments: We thank W. Li and D. Oakley for technical assistance; D. Dolezel for technical advice; the Holy and Taghert laboratories for advice; E. Herzog for comments on the manuscript; the Bloomington Stock Center, Janelia Research Center, J. Kim, and M. Affolter for sharing fly stocks; and M. Rosbash for antibodies to PER. Supported by the Washington University McDonnell Center for Cellular and Molecular Neurobiology and by NIH grants R01 NS068409 and R01 DP1 DA035081 (T.E.H.) and NIMH 2 R01 MH067122-11 (P.H.T.). Author contributions: X.L., T.E.H., and P.H.T. conceived the experiments; X.L. performed and analyzed all experiments; and X.L., T.E.H., and P.H.T. wrote the manuscript. T.E.H. has a patent on OCPI microscopy. Materials are available upon request.

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