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. 2004 Sep 8;24(36):7951-7.
doi: 10.1523/JNEUROSCI.2370-04.2004.

The neuropeptide pigment-dispersing factor coordinates pacemaker interactions in the Drosophila circadian system

Yiing Lin  1 , Gary D Stormo, Paul H Taghert
Affiliations

The neuropeptide pigment-dispersing factor coordinates pacemaker interactions in the Drosophila circadian system

Yiing Lin et al. J Neurosci. .

Abstract

In Drosophila, the neuropeptide pigment-dispersing factor (PDF) is required to maintain behavioral rhythms under constant conditions. To understand how PDF exerts its influence, we performed time-series immunostainings for the PERIOD protein in normal and pdf mutant flies over 9 d of constant conditions. Without pdf, pacemaker neurons that normally express PDF maintained two markers of rhythms: that of PERIOD nuclear translocation and its protein staining intensity. As a group, however, they displayed a gradual dispersion in their phasing of nuclear translocation. A separate group of non-PDF circadian pacemakers also maintained PERIOD nuclear translocation rhythms without pdf but exhibited altered phase and amplitude of PERIOD staining intensity. Therefore, pdf is not required to maintain circadian protein oscillations under constant conditions; however, it is required to coordinate the phase and amplitude of such rhythms among the diverse pacemakers. These observations begin to outline the hierarchy of circadian pacemaker circuitry in the Drosophila brain.

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Figures

Figure 1.
Figure 1.
After light entrainment, dissipation of locomotor rhythms in pdf01 mutants is gradual. Young female flies were entrained for 6 d in 12 hr LD cycles and then released into DD for 9 d. The representative data are double plotted, with the last 24 hr repeated on the next row. Rhythmic (R) and arrhythmic (AR) designations refer to behavioral analysis results from periodogram analysis for DD days 1-9. Most control flies (y w;;) remain rhythmic in DD. pdf01 flies (y w;; pdf01) often retain residual rhythms for 2-3 d of DD before becoming arrhythmic. Approximately 30% of pdf01 flies retain weak, fast-running rhythms in DD (example in the rightmost plot).
Figure 2.
Figure 2.
The phasing of the small LNv neurons disperses over time. On the right, each plot shows the percentage of cells expressing PER in their cytoplasm or nucleus across a circadian cycle from control (y w;;) and pdf01 mutants (y w;; pdf01). The y-axis represents the average percentage of cytoplasmic PER cells in the small LNv cluster of one brain hemisphere. An average of 13 hemispheres are represented in each time point. Error bars indicate SEM. Cells were visible and scored at all time points, except at CT13 of DD6 in both control and pdf01 flies, when low PER immunoreactivity precluded identification of the small LNvs (indicated as breaks in the graph). All plots showed statistically significant rhythms by ANOVA testing (p < 0.05). x-axis represents CT. Markings denote statistically equivalent time points by the Tukey multiple comparison test: boxed time points have peak values, and bracketed time points have trough values. Example images of small LNvs are shown on the left. For both control and mutant genotypes, the neurons displayed nuclear PER at CT5 and CT23 across all days. Control flies display predominantly cytoplasmic PER at CT17, whereas individual brains of pdf01 mutants have mixed cytoplasmic (“doughnuts”) and nuclear distributions of PER at CT17. The overall intensity of these images was adjusted to facilitate viewing of the PER signal.
Figure 3.
Figure 3.
The LNds of pdf01 mutants phase advance in DD. Please refer to Figure 2 for details on figure format. In the seneurons, cytoplasmic PER is seen at CT17 of control flies but earlier in pdf01 mutants. Across all DD days, the LNds have predominantly nuclear PER at CT5 and CT23. Low PER staining in control brains stained at CT13 of DD3 precluded identification of LNds. An average of 13 hemispheres are represented in each time point. All plots showed statistically significant rhythms by ANOVA testing (p < 0.05). An asterisk on the representative image of y w on CT5 of DD9 marks a sinus-like feature, which has not been described previously, around which the LNds were consistently observed.
Figure 4.
Figure 4.
PER intensity rhythms persist in the small LNvs of pdf01 mutants. Confocal images from a subset of brains are shown; quantified intensity values are shown in the graphs to the right. At some time points, PER staining was too low to allow for reliable identification of small LNvs. In these panels, such as CT11 of DD day 3 in control flies, blank squares exist to indicate that fewer than eight examples could be found. On average, 15 brain hemispheres per time point were imaged. All plots showed statistically significant rhythms by ANOVA testing (p < 0.05). In the graphs, the y-axis represents absolute intensity values of a maximum of 4095. x-axis represents CT. Markings denote statistically equivalent time points by the Tukey multiple comparison test: boxed time points have peak values, and bracketed time points have trough values.
Figure 5.
Figure 5.
PER intensity rhythms phase advance and then diminish in the LNds of pdf01 mutants. Please refer to Figure 2 for details on figure format. All plots showed statistically significant rhythms by ANOVA testing (p < 0.05), except for the data from y w;; pdf01, DD day 9.
Figure 6.
Figure 6.
A model for interactions between the two lateral neuron pacemaking centers in the Drosophila brain. In wild-type flies (top), the small LNv center (s-LNv) maintains the normal phase and amplitude of molecular rhythms in the LNd center as well as its own synchronicity through PDF communication (possibly autocrine). Projections from the s-LNvs and/or the LNds regulate premotor centers (PMC) and organize behavioral rhythms. In the absence of PDF (in pdf01 mutants, bottom), a syndrome of effects is observed: the s-LNvs become desynchronized, but because they are effectively silenced by the pdf01 mutation, the effects of this decoupling are unlikely to influence the PMC. With the lack of PDF entrainment, the LNds revert to a low-amplitude, fast-running clock; however, PDF is not required for their synchronization. The loss of a strong circadian signal from the LNd and/or the s-LNv into the PMC leads to behavioral arrhythmicity. Shades of gray represent differences in phase and/or period. The graphs to the left represent the decreased amplitude of PER staining rhythms in LNds and the desynchrony of staining rhythms in the LNvs of pdf01 mutants.
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