Multiple amidated neuropeptides are required for normal circadian locomotor rhythms in Drosophila

Abstract

In Drosophila, the amidated neuropeptide pigment dispersing factor (PDF) is expressed by the ventral subset of lateral pacemaker neurons and is required for circadian locomotor rhythms. Residual rhythmicity in pdf mutants likely reflects the activity of other neurotransmitters. We asked whether other neuropeptides contribute to such auxiliary mechanisms. We used the gal4/UAS system to create mosaics for the neuropeptide amidating enzyme PHM; amidation is a highly specific and widespread modification of secretory peptides in Drosophila. Three different gal4 drivers restricted PHM expression to different numbers of peptidergic neurons. These mosaics displayed aberrant locomotor rhythms to degrees that paralleled the apparent complexity of the spatial patterns. Certain PHM mosaics were less rhythmic than pdf mutants and as severe as per mutants. Additional gal4 elements were added to the weakly rhythmic PHM mosaics. Although adding pdf-gal4 provided only partial improvement, adding the widely expressed tim-gal4 largely restored rhythmicity. These results indicate that, in Drosophila, peptide amidation is required for neuropeptide regulation of behavior. They also support the hypothesis that multiple amidated neuropeptides, acting upstream, downstream, or in parallel to PDF, help organize daily locomotor rhythms.

Fig. 1.

Expression of PHM immunostaining driven by different gal4 drivers in a PHM null background. Each column displays a three-part series of confocal images from a single adult male brain of the genotype indicated. Thebottom panels (Whole) represent overlays of the three-volume sections and depict nearly the entire brain thickness. The three separate scans display anterior,middle, and posterior sections of the brain. For the wild-type (WT) brain,36Y-rescued animals, and c929-rescued animals, section depths were 66, 60, and 60 μm, respectively. For386Y-gal4-rescued animals, section depths were 72, 60, and 60 μm, respectively.

Fig. 2.

Schematic diagrams of the staining patterns displayed in Figure 1. The genotypes are indicated to theleft of each panel. The sizes and positions of various cell bodies are approximate. Black indicates strong staining; gray indicates weak to moderate staining. Many cell types included in these patterns appeared similar; double antibody staining experiments confirmed that many cellular elements are shared (see text for further details). MP1, MP2,MP3, LN, and SE indicate prominent peptidergic cell groups; most preparations included one or more representatives of each group.

Fig. 3.

Double-immunostaining to identify PDF neurons included in gal4 expression patterns. Confocal scans of the brain from an adult PHM mutant animal that was rescued by a combination of two gal4 elements (c929-gal4 and386Y-gal4) driving UAS-PHM. The tissue was stained for PHM and proPDF antibodies. Large LN-Vs are stained by both PHM (A andC,red) and proPDF (B andC, green). Small LN-Vs are only stained by proPDF antibodies (E and F,green), but not at all by PHM antibodies (D and F, red). Note another PHM-positive cell body in the vicinity of the small LN-Vs that is not proPDF-positive (F). These images were taken from different focal planes of the same specimen. Scale bar, 20 μm.

Fig. 4.

Locomotor activity of normal,per⁰, and singlegal4:UAS-PHM-rescued PHMmutant flies. Average activity histograms indicating relative levels of locomotion. White and black bars indicate the day and night phases in LD, respectively (Hamblen-Coyle et al., 1989, 1992). n, number of flies tested. For the constant dark (DD) plots (rows 2 and3), white bars designate the subjective day. Dots indicate SEM values for that 30 min time bin with reference to average level of activity per fly. A,F, K,per⁰¹; B,G, L,36Y-gal4:UAS-PHM-rescuedPHM mosaics; C, H,M,c929-gal4:UAS-PHM-rescuedPHM mosaics; D, I,N,386Y-gal4:UAS-PHM-rescuedPHM mosaics; E, J,O, Canton-S wild type.A–E, 7 d of LD behavior;F–J, behavior during DD days 1–2;K–O, behavior during DD days 3–9.ZT, Zeitgeber time; CT, circadian time.

Fig. 5.

Numerical measures of varying behavioral rhythm strengths in per⁰¹, normal, and singlegal4-rescued PHM mutants. Signal-to-noise ratios (SNRs) for the final 7 d (DD days 3–9) of the free running period (see Table 4). The panelsindicate SNR distributions for per⁰¹,y w;c929-gal4/UAS-PHM,PHM⁰², and Canton-S (top); and y w;PHM⁰¹/UAS-PHM, PHM⁰² containing either36Y-gal4,c929-gal4, or386Y-gal4 (bottom). SNR values ≤1.04 were divided into increments of 0.1; between 1.05 and 2.64, SNR values were divided into increments of 0.3; all SNR values >2.65 were grouped together. The ordinate values are the percentage of total flies whose SNR falls within each interval. Thenumbers of flies that were scored arrhythmic by χ-square periodogram analysis are indicated above the histogram bars. For the rhythmic individuals, free-running periods of the different genotypes were calculated by Maximum Entropy Spectral Analyses independently of those in Table 4; they were not significantly different by ANOVA (means: Canton-S, 24.1 ± 0.2 hr;per⁰¹, 24.9 ± 1.2 hr; y w; PHM⁰¹/UAS-PHM, PHM⁰²;386Y-gal4/+, 23.4 ± 0.3 hr;y w;PHM⁰¹/UAS-PHM, PHM⁰²;36Y-gal4/+, 23.9 ± 0.8 hr; y w;c929-gal4/UAS-PHM,PHM⁰², 23.8 ± 1.1 hr).

Fig. 6.

Locomotor activity of doublegal4:UAS-PHM-rescued PHMmutant flies. Average activity histograms for groups of flies, plotted as described in Figure 4. n, number of flies tested. A, D, G,36Y-gal4/c929-gal4:UAS-PHM-rescuedPHM mosaics; B, E,H,c929-gal4/D42-gal4:UAS-PHM-rescuedPHM mosaics; C, F,I,c929-gal4/pdf(M)-gal4:UAS-PHM-rescuedPHM mosaics.

Fig. 7.

Locomotor activity of doublegal4:UAS-PHM-rescued PHMmutant flies. Average activity histograms for groups of flies, plotted as described in Figure 4. n, number of flies tested.A, D, G,c929-gal4/pdf(N)-gal4:UAS-PHM-rescuedPHM mosaics; B, E,H,c929-gal4/Appl-gal4:UAS-PHM-rescuedPHM mosaics; C, F,I,c929-gal4/c155-gal4:UAS-PHM-rescuedPHM mosaics.

Fig. 8.

Locomotor activity of doublegal4:UAS-PHM-rescued PHMmutant flies. Average activity histograms for groups of flies, plotted as described in Figure 4. n, number of flies tested.A, D, G,36Y-gal4/tim(#16)-gal4:UAS-PHM-rescuedPHM mosaics; B, E,H,c929-gal4/386Y-gal4:UAS-PHM-rescuedPHM mosaics; C, F,I,c929-gal4/tim(#16)-gal4:UAS-PHM-rescuedPHM mosaics.

Fig. 9.

Numerical measures of varying behavioral rhythm strengths in double gal4-rescued PHMmutant flies. SNR values for the final 7 d (DD days 3–9) of the free running period presented as described in Figure 5. Thepanels indicate SNR distributions for y w; PHM⁰¹/UAS-PHM, PHM⁰² containing eitherc929-gal4 and36Y-gal4,c929-gal4 andD42-gal4, orc929-gal4 andpdf(M)-gal4(top); y w;PHM⁰¹/UAS-PHM, PHM⁰² containing eitherc929-gal4 andpdf(N)-gal4,c929-gal4 andAppl-gal4, orc929-gal4 and c155-gal4(middle); and y w;PHM⁰¹/UAS-PHM, PHM⁰² containing eitherc929-gal4 and386Y-gal4,c929-gal4 andtim(#16)-gal4, or36Y-gal4 andtim(#16)-gal4(bottom). The ordinate values are the percentage of total flies whose SNR falls within each interval. Thenumbers of flies that were scored arrhythmic by χ-square periodogram analysis are indicated above the histogram bars. For the rhythmic individuals, free-running periods of the different genotypes were calculated by Maximum Entropy Spectral Analyses, independently of those in Table 4; they were not significantly different by ANOVA (means = y w;c929-gal4,PHM⁰¹/UAS-PHM, PHM⁰²;36Y-gal4/ +, 25.4 ± 1.5 hr;y w, pdf-gal4(M); y w;c929-gal4/UAS-PHM,PHM⁰², 24.4 ± 0.4 hr; y w; c929-gal4,PHM⁰¹/ UAS-PHM, PHM⁰²;D42-gal4/ +, 24.2 ± 1.0 hr;y w, pdf-gal4(N);c929-gal4,PHM⁰¹/ UAS-PHM, PHM⁰², 25.2 ± 0.3 hr; y w; c929-gal4,PHM⁰¹/ UAS-PHM, PHM⁰²;Appl3GK-gal4/ +, 24.0 ± 0.4 hr;y w; c929-gal4 and c155-gal4, 24.1 ± 0.6 hr; y w;c929-gal4,PHM⁰¹/UAS-PHM, PHM⁰²;386Y-gal4/+, 24.1 ± 0.3 hr;y w; c929-gal4,PHM⁰¹/ UAS-PHM, PHM⁰²;tim(#16)-gal4/ +, 24.0 ± 0.2 hr;y w;PHM⁰¹/UAS-PHM, PHM⁰²;tim(#16)-gal4/36Y-gal4, 24.9 ± 0.4 hr).