The Drosophila circadian clock is a variably coupled network of multiple peptidergic units

Abstract

Daily rhythms in behavior emerge from networks of neurons that express molecular clocks. Drosophila's clock neuron network consists of a diversity of cell types, yet is modeled as two hierarchically organized groups, one of which serves as a master pacemaker. Here, we establish that the fly's clock neuron network consists of multiple units of independent neuronal oscillators, each unified by its neuropeptide transmitter and mode of coupling to other units. Our work reveals that the circadian clock neuron network is not orchestrated by a small group of master pacemakers but rather consists of multiple independent oscillators, each of which drives rhythms in activity.

Fig. 1. The PDF positive clock neurons coherently set free-running periods via PDF signaling over a limited temporal range

(A to C) Scatter plots of the predominant free-running periods of rhythmic flies overexpressing different forms of DBT or SGG in both PDF positive and negative clock neurons (driven by Clk-GAL4) (A), and only in the PDF positive neurons (driven by Pdf-GAL4) of wild-type flies (B) or Pdfr^- mutants (C). Circles indicate the highest amplitude free-running period for individual rhythmic flies; lines represent mean ± SEM. DBT^S, DBT^Short; SGG^CA, constitutively active SGG; SGG^WT, wild-type SGG; SGG^Hypo, hypomorphic SGG; SGG^KD, kinase-dead SGG; DBT^WT, wild-type DBT; DBT^L, DBT^Long. Kruskal-Wallis one-way ANOVA reveals a significant difference among groups in (A) and (B) (p < 0.0001 for both), but no significant difference among groups in (C) (p = 0.4829).

Fig. 2. The PDF negative clock neurons exert independent control over free-running activity rhythms

(A and B) Scatter plots of the predominant free-running periods of rhythmic flies overexpressing DBT^S, SGG^Hypo or DBT^L only in the PDF negative neurons (driven by Clk-GAL4/Pdf-GAL80) of wild-type flies (A) or Pdfr^- mutants (B). Kruskal-Wallis one-way ANOVA reveals a significant difference among groups in both (A) and (B) (p < 0.0001 for both). (C) Scatter plots of the predominant free-running periods of rhythmic flies with different compositions of PDF positive and negative clock neurons. Specific genotypes are: per⁺, Pdf>DBT^S for “Fast PDF+, Norm PDF-”, per⁰¹, Pdf>PER for “per⁺ PDF+, per⁰¹ PDF-”, and per⁰¹, Pdf>PER+DBT^S for “Fast PDF+, per⁰¹ PDF-”. (D to F) Representative actograms (upper panels) and χ-square periodograms (lower panels) of individual flies with different compositions of PDF positive and negative neurons under constant darkness. Genotypes are as follows: (D) per⁺, Pdf>DBT^S, (E) per⁰¹, Pdf>PER, and (F) per⁰¹, Pdf>PER+DBT^S.

Fig. 3. PDF modulates only half of the PDF negative dorsal lateral neurons

(A) A representative micrograph showing the dorsal lateral neurons (LN_ds) from a Clk>Epac1-camps fly brain. Four of the six LN_ds (labeled 1-4) were present in the optical section. Scale bar, 5 μm. (B to F) cAMP dynamics of LN_ds in response to bath-applied 10^-5 M PDF peptide (green triangles). Responses of 45 LN_ds imaged from 13 Clk>Epac1-camps brains shown in (B) fell into two classes: responsive LN_ds (22/45) that displayed large cAMP increases (> 10% change in CFP/YFP ratio) (C), and non-responsive LN_ds (23/45) (< 10% changes) (D). The colored traces in (B to D).are from the LN_ds shown in (A) circled with the same color as their plots. All PDFR⁺ LN_ds (18 neurons imaged from 7 Mai179>Epac1-camps brains) displayed significant cAMP increases in response to PDF application (E). None of the PDFR^- LN_ds (11 neurons imaged from 6 Clk/cry-GAL80>Epac1-camps brains) displayed significant cAMP increases (F). (G) Summary of maximum cAMP responses of LN_ds to 10^-5 M PDF. NR: non-responsive LN_ds from (D); R: responsive LN_ds from (C); PDFR⁺ and PDFR^- are from (E) and (F), respectively. The letters ‘a’ and ‘b’ denote significantly different groups (p < 0.0001), by Kruskal-Wallis one-way ANOVA and Dunn’s multiple comparisons test. (H) cAMP responses of LN_ds to bath-applied 10^-5 M forskolin, a direct activator of adenylyl cyclases. “All” represents forskolin responses of LN_ds recorded from Clk>Epac1-camps brains, in which the cAMP sensor was expressed in both PDFR⁺ and PDFR^- LN_ds. The numbers of neurons and brains examined were: All (16, 5), PDFR⁺ (12, 5), and PDFR^- (10, 6). NS: not significant, by Kruskal-Wallis one-way ANOVA and Dunn’s multiple comparisons test. For all histograms, data are presented as mean ± SEM.

Fig. 4. Physiological connectivity does not ensure molecular clock coupling in the lateral neuron network

(A to C) Immunostaining of PER protein in PDF positive and negative lateral neurons across different time-points on day 4 of constant darkness (DD4). In UAS-DBT^L control fly brains, PER accumulated in the PDF positive (s-LN_vs) and negative (LN_ds and 5^th s-LN_v) neurons with the same phase (A). In Pdf>DBT^L flies in which the PDF neurons were slowed down, only two LN_ds (marked by yellow arrows) were coupled with the s-LN_vs, displaying a shifted phase of PER cycling relative to the other LN_ds (B). In Pdfr^-, Pdf>DBT^L flies, all PDF negative neurons (LN_ds and 5^th s-LN_v) had similar phases of PER cycling, and none were coupled to the uniformly delayed s-LN_vs (C). Scale bars, 5 μm. (D to F) Quantification of PER immunostaining intensity within lateral neurons of UAS-DBT^L flies (D), Pdf>DBT^L flies (E), and Pdfr^-, Pdf>DBT^L flies (F). The LN_ds in (E) were divided into two groups based on their phase differences and quantified separately: the two LN_ds coupled to the s-LN_vs were quantified as “LN_d (shifted)” and the others as “LN_d (unshifted)”. (G to J) The two shifted LN_ds express neuropeptide sNPF. LN_ds were co-immunostained for PER and sNPF at CT0 and CT12 on DD4 (CT0 and CT12 correspond to the light-on and light-off times had the 12hr: 12hr light: dark cycles continued). In UAS-DBT^L control flies, the two sNPF⁺ LN_ds and four sNPF^- LN_ds had similar subcellular PER distribution (G) and expression levels (I) at each of these time-points. In Pdf>DBT^L flies, the sNPF⁺ LN_ds differ in their PER distribution (H, yellow arrows) and intensity (J) from the sNPF^- LN_ds. Scale bars, 5 μm. Asterisks in (G) and (H) indicate sNPF⁺ cells that are not clock neurons (lack of PER expression). The letters ‘a’ and ‘b’ in (I) and (J) denote significantly different groups (p < 0.0001 for both), by Kruskal-Wallis one-way ANOVA and Dunn’s multiple comparisons test. Sample sizes are reported in Table S6 for (D to F), and in Table S7 for (I and J).