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. 2008 Nov 4;6(11):e273. doi: 10.1371/journal.pbio.0060273

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© 2008 Wu et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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Flies with pdf-GAL4 and either UAS- δ-ACTX-Hv1a (4×UAS) or UAS-μO-MrVIA transgenes were entrained for at least 4 d in 12-h:12-h LD conditions and then brains were processed for anti-PDF immunofluorescence. Green anti-PDF signal indicates cytoplasmic PDF accumulation in sLN_V dorsomedial terminals.

(A) Average activity histograms indicate relative levels of locomotor activity versus time for flies of the indicated genotypes over 5 d in LD. Each bar represents 30-min cumulative activity, with white and black bars indicating the day and night phases. Flies expressing membrane-tethered δ-ACTX-Hv1a exhibit phase advanced lights-on anticipation as revealed by the change in shape of the envelope of relative activity in the 3 h prior to lights-on from concave to convex with increasing membrane-tethered δ-ACTX-Hv1a expression.

(B) Bar graph shows computed phase scores (mean ± SEM) for lights-on anticipation, defined as relative cumulative activity in the 3 h just before lights-on divided by the 3 h just after lights-on. Indicated genotypes represent flies expressing either membrane-tethered μO-MrVIA or δ-ACTX-Hv1a with one, two, four or six copies of UAS. Membrane-tethered δ-ACTX-Hv1a expression dose-dependently phase advances morning anticipation of lights-on. The difference between control pdf>μO-MrVIA and each of the pdf> δ-ACTX-Hv1a genotypes is statistically significant by ANOVA with Tukey-Kramer multiple comparison (p < 0.001).

(C) Bar graphs show normalized integrated anti-PDF staining intensity (mean ± SEM). Representative immunofluorescence images of sLN_V dorsomedial terminals from flies of the indicated genotypes at the indicated time points are shown. Control flies expressing membrane-tethered μO-MrVIA exhibit peak PDF accumulation at ZT2–ZT6 and trough at ZT14–ZT18 (p < 0.001). In contrast, membrane-tethered δ-ACTX-Hv1a–expressing flies exhibit peak PDF accumulation at ZT22 and trough at ZT10–ZT14 (p < 0.001), representing an approximately 4-h phase advance of the rhythm of PDF accumulation. The differences among different genotypes at different circadian times were compared with ANOVA and Tukey-Kramer multiple comparisons. n > 22 brain hemispheres for each experimental group.